Thursday, June 30, 2016

BtB#7: Immune cells of Central Nervous System


In a blog post long ago, I had commented that the central nervous system is immunologically isolated from rest of the body. In the post, I mainly focussed on talking about the need for an isolated system. In the event of inflammation and breach of BBB (Blood Brain Barrier), many different peripheral immune cells are allowed access to the brain. Under normal circumstances, most of the immunological functions are managed by cells exclusive to CNS. Very recently, it has been shown that CNS has a lymphatic system of its own. That means a lot of things that we assumed about CNS immunology is wrong and new research will be needed to rectify older theories.

Fig 1: Cells of CNS.
The CNS cells are divisible to into 2 different basic types- Neuronal cells and Glial cells. Glia refers to "cementing material". Earlier it was thought that glial cells are simply supporting cells and doesn't have any much function. Studies have proven otherwise. The glial cells are classified into 3 types
  • Microglia
  • Macroglia
  • Others

Of all the cells, Microglia is considered as the innate immune cell and first and line of defence. Astrocytes were thought of as a cell of importance in neuronal regulation. However, most recent findings conclude that they are important in regulating microglia and function as an immune modulator. Thus they too are included as Immune cells (at least in part) of CNS.


Fig 3: Structure of Microglia.
Microglia is originally derived from yolk sac during the developmental stage and is exclusively maintained in CNS. In the case of depletion of the microglia, they can be derived from peripheral myeloid cells. The microglia forms roughly 10–15% of all cells found within the brain. They can be identified by flow cytometry using phenotype CD11b+, CD45low.

Microglia in the brain exists in 2 possible forms- (i) Ramified or (ii) Activated. Everything described in the literature is one of the 2 forms or an intermediate state.

Table 1: Factors involved in activation of Microglia.
Ramified microglia are simply resting microglial cells. They are identified by their long thin branching processes and a small cellular body. It has been noted that each microglia has a micro-territory where it works and scans for damage. They are immunologically inactive (They possess very low MHC I/II molecules on their surface). The branching process moves very rapidly around the environment (estimated speed of 1.5 µm/min). These cells are extremely sensitive to changes in the environment. Microglia can be activated several factors. The factors are categorised into 2 types- "Off signal" and "On signal". Other than the innate molecules molecules such as LPS and toxins can activate microglia. In the event of a neural damage, the microglial cells are rapidly activated. The cells become motile, and using amoeboid-like movements and immediately surround the site of damage. If the damage persists and cells begin to die or degenerate microglial cells undergo further differentiation and assume the role of phagocytes.

Fig 4: Microglial functions. Source
Microglia offers several functions in CNS such as scavenging, phagocytosis, neuronal repair. Even on a day to day basis, several types of cellular debris accumulates in the brain. These are cleared up the microglial cells. One important function is neuronal repair. After an inflammatory event the microglia clears up the cellular mess and induces neuronal repair. This is achieved by secreting anti inflammatory cytokines, recrutiment of astroglial cells and promoting building of neuronal cell network. This is achieved through a mechasnim called as synaptic stripping. Microglia physically interacts with injured neurons and remove synapses which can extend to removing entire dendritic trees (depending on extent of damage). This allows proper reforming of new synapse and connections.

Microglia via its interaction with neuronal cells is recognised to play inportant roles in Mental health, memory formation and neurogenesis. Microglial dysregulation is considered as one of the important factors involved in Behavioral disorders and conditions such as Alzheimers (Lack of phagocytic activity leading to accumulation of Aβ Proteins).

Fig 5: CD4+ T cells interaction with microglia. Source
Just like Monocytes, and macrophages, microglia can have a range of phenotypes depending on their microenvironment. These are refferred as polarisation states. Microglia polarization states can be one of the following- classical activation (M1), alternative activation (M2a), type II alternative activation (M2b) or acquired deactivation (M2c). This polarisation can be achieved through interaction with different types of molecules or cells. For example, interaction with Th1 and Th17 cells leads to a more M1 like state. Th2 and Treg can influence M2 like. See Fig 5.


Fig 6: Neuron-Microglia and Astrocyte
Microglia Signaling Pathways. Source
As already explained astrocytes are not classicaly considered as immune cells. More recently it has however become clear that astrocytes control the microglial activity and the reverse is also true. It's a two way signalling system and both of them are also in direct contact with neurons.

Astrocytes form roughly about 40% of the glial cells and are the main supporting cells of Neurons. They are derived from the neuroepithelial cells during development. Their major role in structural (supporting neurons, formation of BBB), metabolic support for neurons (Regulation of ion concentration, fuel supply) and modulate synaptic transmission.

Immunologically, Microglia constantly communicates with astroglia via a 2 way signal. For example, Microgllia can be activated by ROS/RNS which can be controlled by Astroglia through soluble anti oxidant factors (thus controlling microglia activation). Another example, astroglia constantly removes K+ ions which otherwise leads to activation of microglia.

Table 2: Astrocyte-produced molecules and
immune functions. Source
In the event of chronic microglia activation, microglia can produce excessive quantitites of TNF-α. The excessive TNF-α then dampens the ability of astrocyte to uptake glutamate thereby leading to accumulation and neuronal toxicity (Excitotoxicity). I'm basically illutsrating some examples to imply that astrocytes and microglia immunologically talk to each other. Astrocytes can produce a variety of cytokines which mediates a variety of effects. Table 2 is a summation of molecules that exert or regulate pro-inflammatory functions.

To summarise, microglia are the primary immune cells of the CNS regulated mainly by astroglial cells. I really like this idea, "If Microglia is the police, Astroglia polices the police". Microglia and astroglia manages the immunology show within CNS, and only when help is needed does it allow peripheral cells to further participate.

      Louveau, Antoine et al. "Structural And Functional Features Of Central Nervous System Lymphatic Vessels". Nature 523.7560 (2015): 337-341.

Derecki, Noël C., James C. Cronk, and Jonathan Kipnis. "The Role Of Microglia In Brain Maintenance: Implications For Rett Syndrome". Trends in Immunology 34.3 (2013): 144-150.

Sofroniew, Michael V. "Astrocyte Barriers To Neurotoxic Inflammation". Nature Reviews Neuroscience 16.5 (2015): 249-263.

Shih, A. Y. "Policing The Police: Astrocytes Modulate Microglial Activation". Journal of Neuroscience 26.15 (2006): 3887-3888.

Monday, June 27, 2016

HIV nuclear entry


HIV or Human immunodeficiency virus is a topic that I have blogged multiple times. Every week I see so many publications about HIV. It is one of the absolutely highly researched topics in infectious diseases and yet we neither have a cure nor have a vaccine. Retroviruses in comparison to other groups of viruses have one special property. They are made up of RNA, gets converted to DNA and then integrate with the genome. But what is special to HIV is the way it does it. Simple lentiviral members like MLV (Murine Leukemia Virus) rely on cell division process to integrate their genome to the host. During the cell division process, the nucleus opens and virus gains entrance to the nuclear material. But HIV simply makes its way in through nuclear transport.

Fig 1: HIV Life Cycle.
Nuclear transport of HIV is a well-studied phenomenon. The Reverse transcriptase has three domains: thumb, palm, finger and RNAaseH. This enzyme uses one RNA copy from the HIV genome to make single stranded DNA by reverse transcription and then double-stranded DNA from it. The RNA is degraded by RNAaseH. The presence of 2 copies of RNA facilitates floppy replication and enhances replication errors. The formation of dsDNA continues in the Reverse Transcriptase Complex (RTC), simultaneously migrating towards the nucleus using microtubule network with a cellular dynein-dependent motor complex. On the formation of DNA, the complex is referred to as the Pre-Integration Complex (PIC).

Fig 2: Structure of Nuclear Pore.
PIC interacts with several different factors that influence its nuclear import. It is currently not clear as to what are the factors important for nuclear factors. Though many of them have been identified which includes TNPO3, NUP358, NUP153, Integrase, LEDGF, CypA, CPSF6 and much more. There is one fundamental problem in understanding the nuclear import of HIV in all the studies. HIV is transported to nucleus through nuclear import factors by passing through the pore. However, the nuclear pore is much smaller than the HIV PIC. So how does it still manage to pass? 

Let me digress a little bit. The nucleus of a cell is not accessible to chemicals for direct communication. The Nucleus is well guarded and specific channels are provided in the membrane called as Pores. The nuclear pore is a protein-lined channel in the nuclear envelope that regulates the transportation of molecules between the nucleus and the cytoplasm.

Fig 3: Model of KIF5B mediated
nuclear import of HIV-1. Source
In a study published by Edward et al; they looked into this question. In the first part of the study, they found that depletion of KIF5B (Kinesin Family Member 5B) and Nup358 (nucleoporin 358) led to the accumulation of viral cores around the nucleus, with a reduction in nuclear import. This is consistent with previous reports indicating that these are required for nuclear import of HIV. Subsequent investigation showed that HIV-1 induces NUP358 relocalization in a KIF5B dependent manner. The last part of the experiments yielded that KIF5B transported Nup358 away from nucleus thus making the pore wide enough for HIV to pass through. It is essentially tearing of the nuclear membrane. A model of how KIF5B is involved in HIV nuclear import is shown in Fig 3. The authors also noted based on previous studies that failing to import (which means the HIV is staying in the cytoplasm for a longer time), activate cytoplasmic host cell sensors and initiate interferon response.

Based on the study it has been suggested that blocking KIF5B-HIV interaction should be able to block the nuclear import and thus give more time for the cell to sense and develop a response. As one of the author states, "It's like making a bank vault harder to break into. In addition to making the money more secure, it would increase the chance of sounding the alarm and catching the burglars."

Thursday, June 23, 2016

NGS for Microbial Diagnostics


I have multiple times talked about current lacunae in culture based identification of bacteria from clinical samples. As much as is the problem of time involved in classical methods, there is equally a problem in finding the culprit. In an ideal scenario, Microbial diagnostics should be able to catch up the etiological agent by growing them on a media and then subsequently identifying them. In reality, most of the labs are far from ideal. Studies indicate that often microbial identification is missed because there is too much reliance on culture.

Long ago I had written a post for why bacterial growth in culture is not as simple as it seems to be (Link). Common media used in the laboratory include Blood agar, MacConkey agar, Nutrient agar, CLED agar etc. Most of these support growth for a limited number of organisms. Let us take an example. Neisseria meningitidis is a well known bacteria involved with meningitis. The growth of this organism has very specific requirements. Unless a clinical suspicion has been raised, specialised media mayn't be used. Occassionally despite extensive workup finding a pathogen may turn out to be difficult.

One of the best example that I recall is a case published in NEJM. The case is that of a 14 year old boy. The diagnostic workup of the case was not yielding. A brain biopsy also didnt reveal anything significant. When the sample was tested by whole genome sequencing Leptospira infection was suspected based on finding 0.016% corresponding read. Later, further molecular work confirmed that the patient had Leptospira santarosai infection. Similar studies have been done earlier. Studies have tried to examine if they could find evidence for infection using 16s rRNA PCR and sequencing in clinical samples. The studies indicate microbiologists are missing a significant lot of cases. It is my understanding from the reports that the microbial identification rate is anywhere between 18 to 40% depending on the techniques used for identification.

In a recent pilot study paper from Johns Hopkins, showed that using Next Generation sequencing technologies and rigourous bioinformatics, organisms can be identified from clinical samples of brain infection. Pardo-Villamizar says, "By incorporating modern genetic sequencing techniques into pathology diagnostics, we were able to investigate the potential presence of infection in 10 subjects and found appropriate explanations of clinical problems in eight out of 10 patient cases examined in this study. We hope to develop this technique further as a way to bring the diagnosis rate of inflammatory brain disorders and infections closer to 100 percent so we can treat patients more effectively." The study first appeared in bioRxiv.

Fig 1: Microbial identification from study. Source
Next generation sequencing or what is now commonly referred to as NGS has rapidly improved our ability to sequence. Read my earlier post. Link

In the study, the number of bacterial and viral reads varied highly (20 to 25,000). By comparing the sequences to databases identification was possible for infectious agent in 3 out of 10 patients. A summary of findings is shown in Fig 1.

Of the cases as mentioned, 3 were highly significant. Pt-8 was from a 67-year-old woman with osteomyelitis and lung disease presented with multifocal brain and spinal lesions. The sequencing from samle yielded 15 reads. When the data was compared only against M tuberculosis genome, 34 reads could be randomly assigned to TB. Patient responded well to anti-TB drugs. Pt-5 was diagnosed of JCV and Pt-10 of EBV. In other cases it was not absolutely ascertainable as to if the detection was clinically relevant or not. 

The study also mentioned that they could identify many different microbial reads which can complicate the findings. For example, Propionibacterium acnes was detected in all samples, indicating microbial spillover from the intervening tissues probably from collection procedure. The authors also acknowledge that such methods are limited by available database and their completeness. As Steven Salzberg comments, "One of the limitations of using this kind of diagnostic tool is that we can only identify the pathogens for which we have the genetic sequences available. As we continue to sequence the genomes of more organisms, the tool will become steadily more powerful."

Of course this is not the first study to look at NGS as a method for Microbial diagnostics. But this study comprehensively captures the data of prospectives that we expect in future. I also understand that the scenario is quite easy when it comes to samples such as blood or CNS samples where the microbial identification is easier. But what about samples such as Urine or skin. Sequencing will complicate the data and it would be a lot harder to understand which sequence is relevant since they have a natural microbial load.

Wilson M, Naccache S, Samayoa E, Biagtan M, Bashir H, Yu G et al. Actionable Diagnosis of Neuroleptospirosis by Next-Generation Sequencing. New England Journal of Medicine. 2014;370(25):2408-2417. DOI: 10.1056/NEJMoa1401268

Köser C, Ellington M, Cartwright E, Gillespie S, Brown N, Farrington M et al. Routine Use of Microbial Whole Genome Sequencing in Diagnostic and Public Health Microbiology. PLoS Pathogens. 2012;8(8):e1002824.

Salzberg S, Breitwieser F, Kumar A, Hao H, Burger P, Rodriguez F et al. Next-generation sequencing in neuropathologic diagnosis of infections of the nervous system. Neurology - Neuroimmunology Neuroinflammation. 2016;3(4):e251. doi: http:/​/​dx.​doi.​org/​10.​1212/​NXI.​0000000000000251

Wednesday, June 15, 2016

VDPV in Sewer sample- Implications


Photo: Poster of Polio-free India.
Polio eradication program is one of the recent successful achievements of Health organisations across many countries including India. The last case of polio was diagnosed in India in January 2011. After maintaining a polio status for a couple of years an independent panel of 11 experts from the South-East Asia Regional Certification Commission for Polio Eradication (SEA-RCCPE) met for two days and reviewed evidence from 11 countries including India. Subsequently, India was declared as a Polio free country.

There are a couple of anticipated problems in all countries currently titled "Polio free". First, Vaccine strains and vaccine derived strains (Reverting mutants) are expected to circulate and hence vaccination has to be continued. Second, there are still regions where polio has not been eradicated and hence strains from these regions can possibly spill and bring back the infection again. Let us have a look at both. 

Table 1: Salk vs Sabin Vaccine for Polio.
Oral polio vaccine contains attenuated virus, capable of activating an immune response in the body. Being a live virus, the strains replicate in the intestine. During this time, the vaccine strain is also excreted. This excreted strain spreads in the local community which can further help in building herd immunity. OPV provides systemic and mucosal immunity for an individual and also intensifies herd immunity it is the preferred vaccine for vaccinating the masses at risk. However, there are rare events where the excreted virus mutates and reverts to its virulent form. These are known as known as a circulating vaccine-derived poliovirus (cVDPV).When a sufficient number of people are immunised by OPV there rare possible event of reverting mutant becomes a significant risk and hence switching to IPV (Injectable polio vaccine, Dead strain) is more useful. This also ensures that immunity continues to be kept against a very rare possible case of vaccine derived polio. Since 2000, more than 10 billion doses of OPV have been administered to nearly 3 billion children worldwide. As a result, more than 13 million cases of polio have been prevented, and the disease has been reduced by more than 99%. During that time, 24 cVDPV outbreaks occurred in 21 countries, resulting in fewer than 760 VDPV cases.

One of the best discussed cases of lack of vaccination is that of Pakistan. Pakistan has still a good number of unvaccinated people against polio. Despite heroic attempts by the workers to vaccinate there has been fierce resistance. On 20 April 2016, polio workers and the police guards were shot dead in Karachi. Polio workers in Pakistan are often targeted by militant groups due to several misconceptions. Events such as these make polio vaccination has been a huge challenge in Pakistan. In 2015, Pakistan reported 54 of 74 worldwide cases of polio. The implication being that Pakistan is geographically near to India and spillover of the virus can happen at any time if sufficient vaccination has not been done.

Polio P2 Strain found in Hyderabad;
Highlights. Source
That is a good background to start talking about the implications of finding a circulating polio strain in Hyderabad making headlines everywhere. In a routine survey, Vaccine derived polio virus type-II (P2) was found in the lab tests conducted on samples of sewerage water collected at Amberpet in Telangana. Similar situations have been reported earlier in other countries, but this is a first in India after achieving polio-free status. As I already mentioned previously, very rarely VDPV cases do occur. As a preventive measure, the government has decided to vaccinate more than three lakh children in Hyderabad and Ranga Reddy district as a preventive measure. Rajeshwar Tiwari, principal secretary, health states "The government has decided to conduct a special campaign against polio in Hyderabad and parts of Ranga Reddy from June 20 to June 26."

The finding that circulating Poliovirus could be seen indicates that virus can be seen in a community even after immunisation. However, vaccination ensures that people are protected by neutralising antibodies. Countries like US have switched to IPV and doesn't survey for Polio strains in the environment. Experts suggest that circulating strains are unavoidable as long as OPV is in use and not all countries are yet ready to switch to IPV. Multiple publications have indicated that immunisation with IPV will not lead to the eradication of wild-type poliovirus. In order to achieve complete eradication OPV should continue till every country is polio case free then switch to IPV. How long should the IPV be in use is not a question that can be answered currently. India switching to IPV is currently not a good idea since our geographical neighbour is not yet polio free.

In short, finding some Polio strains in environmental samples is not a new deal and there is nothing fancy about it. But it does suggest that we cannot yet let our guard down and must continue  vaccination for at least a few more years. I strongly recommend readers to read Vincent's blog post on Implications of finding poliovirus in sewers. Similar principles apply to our current scenario.


Merten M (2016). India, Pakistan, and polio. BMJ (Clinical research ed.), 353 PMID: 27146842

Duintjer Tebbens RJ, Hampton LM, & Thompson KM (2016). Implementation of coordinated global serotype 2 oral poliovirus vaccine cessation: risks of inadvertent trivalent oral poliovirus vaccine use. BMC infectious diseases, 16 (1) PMID: 27246198

Monday, June 13, 2016

Lab series# 13: Mass Spectrometry Instrumentation Principles


In an earlier post, I had talked about a method called MALDI-TOF (A method in Mass spectrometry) which is making its way into the Microbial diagnostics. The post was more concentrated on my experience with the technique and how it aids microbial identification. Though I talked a little bit about the basics of Mass Spectrometry it was not elaborate. In this post, let us explore Mass Spectrometry instrumentation in detail

Table 1: Common applications of MS. Source
Mass Spectrometry (MS) is an analytical technique, for analysis of chemicals through ionisation and sorting via mass to charge ratio. Every individual chemical is different. Every chemical has a combination of mass and charge ratio that is absolutely unique to it.

These days when you say the word "Mass spec", it largely indicates Proteomics. But that is not true. Any chemical that can be charged and has got a mass can be analysed on a mass spec. Table 1, shows a list of applications that MS can achieve.

MS is actually a method to calculate mass to charge ratios (m/z) and relative abundances for any given molecule. In this case, m stands for mass and z stand for charge number of ions. The number of electrons removed is known as the charge number. m/z represents mass divided by charge number and the horizontal axis in a mass spectrum is expressed in units of m/z. Consider you have two types of balls one heavier and the other lighter. Assume that the balls are travelling at a speed and you have a strong air flowing in a perpendicular path. The lighter is the ball, easier is it to deflect its path. In essence, if you know the original path, speed and the force used for deflection, you can calculate the mass of the ball. Now scale the whole process to an atomic level in a vacuum (10-6 to 10-8 torr) to avoid a molecular collision. That's pretty much how a classic MS works.

Fig 1: Construct of a Mass Spectrometer.
In a classic MS instrument, the process sequence of analysis works in 4 stages.

Stage 1: Ionisation

The atom is ionised by knocking one or more electrons off to give a positive ion. This principle is true for all chemicals including inert materials. Knocking off an electron effectively removes 1 unit of negative charge and hence the molecule is effectively rendered positive charge. Mass spectrometers always work with positive ions. In a majority of case, molecule holds a charge of +1, since it is very difficult to knock off the second electron from an already positively charged molecule.

Stage 2: Acceleration

The ions are accelerated so that they all have the same kinetic energy. This step gives equal chance to all the molecules to fly into the detector and hence their arrival in the detector is now based solely on their mass-charge ratio.

Stage 3: Deflection

The ions are deflected by a magnetic field according to their masses. The degree of deflection depends on the amount of positive charge the molecule is holding and the mass (As already explained).

Stage 4: Detection

The beam of ions passing through the machine is detected electrically, converted to a digital signal and independently analysed by a software and presented in the form of a data.

From the perspective of functional process, Modern MS operates at 3 functional levels, handled by 3 different components.
Fig 2: Basic components of MS.
  • Ion source- Responsible for Ionisation of the molecule
  • Mass Analyser- Responsible for separation of ions based on their mass-to-charge ratio
  • Ion detector- Takes care of detecting and amplifying the signal
The combination of Ion source and Mass analyser determines what type of MS we are talking about. For example- MALDI-TOF is a system where MALDI is the Ionisation source and TOF is the Mass analyser.

Methods in Ionisation:

Table 2: Details of a variety of Ionisation process used for MS.
Ionisation is the process of removing an electron from the molecule rendering it positively charged. The actual technique of ionisation to be employed depends on what is to be ionised. For example, biological specimens and proteins are preferentially ionised by MALDI or ESI. The method of ionisation can be classified into 2 types- Hard and Soft. Hard ionisation techniques are processes which impart high quantities of residual energy in the subject molecule thereby involves large degrees of fragmentation of the molecule. In contrast, soft ionisation imparts little residual energy onto the molecule and therefore result in lower fragmentation. Each method has its own applicability, advantages and disadvantages. See Table 2 for summarised details of most common methods.

Types of Mass Analysers (MA):

All types of MA are based on strong electromagnetic forces applied on the charged Ions. The separation is guided by Lorentz force law and Newton's second law. The acceleration is mass- dependent and dependent on the ionic charge. Therefore, separation of ions will be based on to their m/z value rather than by their mass alone. However, as already discussed in a majority of cases the z=1 and hence separation is based on mass.

Mass analysers are basically divisible into 2 different types- (i) Scanning Ion Monitoring (ii) Selected Ion Monitoring. In scanning ion versions (Ex: TOF) all Ions are studied and analysed whereas in selected ion version (Ex: Magnetic sector and quadrupole mass spectrometers) data is collected only for the masses of interest for specific compounds thereby improving sensitivity. See Table 3, for details of some of the most commonly used Mass analysers.

Table 3: Features of most commonly used Mass analysers

Amongst all of the above, Trapped- Ion MA (More commonly known as Ion traps) are the best and merits some additional discussion. An Ion trap is defined as "Combination of electric or magnetic fields used to capture charged particles, in a system isolated from an external environment." Based on the physics behind the methodology there are different types of traps- Penning trap, Paul trap and Kingdon trap.

The most talked about technology- The Orbitrap is a type of Kingdon trap. Kingdon trap was developed by KH Kingdon. The system consists of a cylindrical outer electrode and a thin wire centre electrode.

Fig 3: Orbitrap analyser. Source
An orbitrap consists of an outer barrel-like electrode and a coaxial inner spindle-like electrode that traps ions in an orbital motion around the spindle. The ions are trapped in an electrostatic field, with central electrode kept at a high voltage. The image current from the trapped ions is detected and converted to a mass spectrum using the Fourier transformation of the frequency signal.

However, injecting the Ions to the trap in itself poses with certain challenges. The problems are overcome by using a separate injection device called as C-trap (curved linear trap for ion injection). The C-trap basically packets the Ions together and injects it into the Orbitrap in batches. Each packet contains a multitude of ions of different velocities spread over a certain volume, therefore, ions will move with different rotational frequencies but with the same axial frequencies. Figure 3 shows a Cross section of the C-trap ion accumulation device and the Orbitrap mass analyser with an example of an ion trajectory. During the voltage ramp, the ion packets enter the Orbitrap mass analyser forming rings that induce current which is detected by the amplifier.

Ion Detectors

Once the molecules are spatially separated based on the m/z ratio, the molecule strikes on the detector. The Ions are electrically detected but usually the signal is extremely weak to make a meaningful data. The function of the detector thus is also to amplify the signal. An ideal detector would have very high amplification, rapid response and a very low noise. There are several types of detector available in the market. By far, 3 types of detector have been used commonly- Electron multipliers, Faraday Cups and Microchannel plates.

Fig 4: Schematics of the two main types
of electron multiplier. Source
Electron Multipliers are useful when the Ion current detected is in the range less than 10-15 amp. When a primary ion strikes a suitable surface (such as that made from CsSb, GaP or BeO) secondary electrons are released. These electrons further stimulate next set of electrons leading to essential amplification. An electrode which emits additional electrons in a photomultiplier or similar amplifying device is called as Dynode. There are 2 subtypes of this design
  1. Discrete dynode electron multiplier
  2. Continous dynode electron multiplier
Fig 5: A schematic diagram of a Faraday cup ion detector.
A Faraday cup (Also known as cylinder electrode detector) is a metal (conductive) cup designed to catch charged particles in vacuum. The resulting current can be measured and used to determine the number of ions or electrons hitting the cup. The Faraday cup is a relatively insensitive detector in comparison to Electron multipliers but is very robust. It is ideally suited for isotope analysis where the signal is higher.

Fig 6: Illustration of microchannel plate. Source
Microchannel plates (MCP) is a planar design made from highly resistive materials (approx 2mm thick) with a regular array of microchannels embedded into it. The microchannels typically measure 10 μm in diameter and spaced apart by 15 μm. In this case, each microchannel acts as a continuous dynode electron multiplier.

Tandem MS or MS/MS

Fig 7: Illustration of Tandem MS. Source
Mass spectrometry is inherently designed to detect and analyse multiple sets of molecules in a rapid time. However, complex samples (most biological samples) are difficult to be analysed with high depth. This problem can be overcome by using an approach called as Tandem MS. Tandem MS represents 2 MS integrated together to perform a very detailed analysis. For example in Tandem MS for protein analysis, peptide ions are formed in the ion source and separated by m/z ratio in the first stage of mass spectrometry. The data is represented as MS1. The peptide Ions breaks into component amino acids through collision-induced dissociation (or other methods) which is separated and detected in the second stage of MS (Data available as MS2). An example schematic of Tandem MS is shown in Fig 7.

Sample injection System for Mass Spectrometry analysis:

When dealing with the analysis of a complex mixture of analytes, introducing all the analytes at once is a problematic situation. There is an inherent limit to how much data can be acquired in a given time for any MS run. For a complex sample, there are too many ions to be detected and analysed. Of course, use of tandem design reduces the problem but there had still been too much complexity. Thus, instead of introducing the whole sample, analytes can be introduced part by part. Let me try an example. Let's say you are analysing a tissue containing about 4000 proteins. There are some abundant proteins which forms a major chunk of protein percentage. Since they are too many in number introducing everything in a single time, the data will capture mostly the major ones. If we spatially separate the sample in such a way that we inject say about 10-20 proteins at a time all of them will be detected. The exact number depends on a variety of factors based on machine design.

Though sample injection is not actually a part of MS, its working is so important that sample injection is considered as a part of MS design. Most modern MS is integrated with HPLC. HPLC-MS/MS (These days they are more commonly known as LC-MS/MS) combines the physical separation capabilities of liquid chromatography with the tandem mass analysis capabilities of MS. In proteomics, chromatographic separation beforehand gives an edge in an analysis. For example, Isomers of a molecule have the same mass and thus multiple isomers may give same MS readout. With HPLC these can be separated before introducing it into MS thus sufficiently differentiating the types. Another idea is to get over the problem of a phenomenon called as "Ion suppression". When a poorly ionised analyte is present in a mixture containing a large amount of something else (such as a buffer), the analysis is significantly affected. Chromatography can largely overcome this issue though cannot be absolutely eliminated.

To summarise working of an MS in a single line, protein preparation is separated using methods such as HPLC which is fed into the Ion source, Ionise, separate the molecules based on m/z value and be detected using detector.

I have tried to limit myself to talking about the basics of MS working without getting into the physics of it. I have also tried to avoid extreme details of working and hence have purposefully omitted several things in the post. In a future post, I will come back with details of how MS is used for proteomics.
Julia H. Jungmann, & Ron M. A. Heeren (2013). Emerging Technologies in Mass Spectrometry Imaging Journal of Proteomics, 75, 16, 5077-5092, 2012 arXiv: 1305.5433v1

Friday, June 10, 2016

Lab Series# 12: Cryo-Preservation of cells


In an earlier post on PBMC isolation technique, I had talked briefly mentioned about cryopreserving cells for downstream processing. I kept the explanations in brief since the focus was on how to get a PBMC from whole blood samples. In this post, I will focus on details of cryopreservation of cells.

Cells have a basic metabolism requirement for survival and hence, all alive cells are metabolically active (By definition). However, when outside the body cells will exhaust their metabolic capability in relatively small time unless continuously maintained in a culture system. Practically a difficult process and then there is a problem of deviation from original properties of the cell to be studied. Cryopreservation provides a suitable alternative by storing the cells in revivable format. The very low temperatures reduce the metabolism rate to near zero.

Cryopreservation is defined as "Use of very low temperatures to preserve structurally intact living cells and tissues". There are several methods defined in the literature, describing the process of cooling the cells. The most well known among them is "Slow cooling technique" and second is a rapid method called "Vitrification". Vitrification is a cryopreservation technique that leads to a glass-like solidification of cells. Oocyte, zygote, embryo and blastocyst freezing by vitrification method for cryopreservation have been used for many years beside sperms preservation. Here I'm restricting the descriptions to slow cooling method. 

Table 1: Comparison of ultra-low temperature storage
methods for cell lines. Source
The process of cooling by itself can be lethal to the cells. There are arguments over what exactly causes cell damage. One theory suggests that the slow freezing causes cellular water to convert to ice crystals which damage the cell structure. Cryoprotectants are supposed to act by reducing the formation of Ice crystals. For biological applications, a cryoprotectant must be able to penetrate cells and have low toxicity. Compounds with such properties include glycerol, dimethyl sulfoxide, ethanediol, and propanediol. In simplest terms, cryopreservation enables cells to be stored thus avoiding the need to have all cell lines in a culture at all times. This drastically enhances the lifespan of a cell. In addition following advantages are offered.
  • Low risk of microbial contamination and cross contamination
  • Avoiding genetic drift and morphological changes 
  • Reduced costs
There are several factors that affect cell viability in cryopreservation. But the most important determining factor is the type of cell to be preserved. Some cells are particularly hard and resistant whereas certain cells are highly sensitive to changes.

The Intracellular water content is dependent on the solute concentration of cells. Anything that effects this solute concentration thus also affects the Ice crystal forming properties. It is known for a long time that the rate of change of temperature controls the transport of water across the cell membrane. The solute in the environment of the cell also contributes to this factor. Together they indirectly influence the probability of intracellular freezing and formation of ice crystals. So if we can control the cellular water levels or external osmotic balance we can avoid ice crystal formation.

Fig 1: Survival of three different cell types.
If the water permeability of the cell membrane and the temperature coefficient of water permeability is known, then it is possible to predict the effect of cooling rate on cell survival. In most cases intracellular freezing is unlikely at 1°C/min. Fig 1, depicts Survival of three different cell types frozen at various cooling rates in 1M DMSO. The figure illustrates that the cooling rate is not a universal value, rather depends on the cell type.

Table 2: CPA classification.
Cryoprotective agents (CPA) are divided into two classes- Intracellular agents and Extracellular agents. A summary of the classification and description is shown in Table 2. In general, best freezing is obtained with a combination of both types. For example in Storing PBMCs, Ficoll remnants act as Extracellular agent and DMSO is used as an Intracellular agent.

There is always some loss of cell viability after cryopreservation. Research is on to identify better CPA's. The new generation of chemicals such as Polyampholytes has shown superior properties in comparison to conventional CPA's (Glycol derivatives). Researchers have also studied Proteins found in cold regions (Commonly known as Antifreeze proteins) for their ability to preserve cells in cold temperatures.

John G. Day, & Glyn N. Stacey (2007). Cryopreservation and Freeze-Drying Protocols Humana Press. Methods in molecular biology. ISBN: 978-1-59745-362-2

Monday, June 06, 2016

Totally Drug Resistant Tuberculosis is actually XDR Plus


In my previous blog post on MCR-1 Positive E coli , I had commented that Total drug resistant TB (TDR-TB), is a misnomer and there probably has never been a case reported as true TDR. I have also argued over why this is so. But, it appears that quite a lot of the readers have a competing opinion. I had a couple of Email's with good arguments and been referred to a paper by Hoffner et al; 2009. There are several pages in WHO site explaining that TDR-TB is not the right term and hence I don't need to talk about it once again. For people who are not aware of TDR Story, here is a snap look.

The First two cases were reported from Italy in 2007. The first female case, Case 1 had acquired TB from her mother (Who was diagnosed with MDR-TB). Case 1 was admitted to three different hospitals for a total of 422 days. Case 1 was admitted to three different hospitals for a total of 422 days. The second female case, Case 2 was admitted to two different hospitals for a total of 625 days. Both died in 2003, after multiple, unsuccessful treatment with all possible Anti-TB available drugs without achieving any significant bacteriological conversion. In both the cases, resistance to new TB drugs had been acquired over a very long time and attributed to inappropriate TB regimen.

In 2007, US reported the first case of XXDR TB from a young Peruvian man, Oswaldo Juarez who was visiting the United States. After his illness puzzled the doctors he visited he was sent to A.G. Holley State hospital. He was treated with high doses of drugs, which are not normally used for TB. He achieved complete cure in 19 months.

Table 1: Patient and Isolate profiles of XXDR TB Isolates.
Table Modified from Original Source
Next report came in 2009 from Iran and Afghanistan. The study characterised a total of 146 MDR-TB strains. Of these, they found 8 isolates were XDR and 15 strains were TDR isolates (10.3%) were identified. The detailed summary of TDR (XXDR) is shown in Table 1.

The next set of reports came from Hinduja Hospital, India. In December 2011, Dr. Udwadia reported four tuberculosis patients resistant to all first and second line TB drugs. By early January, 12 cases were known. As per the Ministry press release then, "The cases reported by Hinduja hospital fall only within the category of 'extensively drug resistant' TB based on standard World Health Organisation (WHO) definitions and not at all as 'totally drug resistant' TB. Of the 12 patients, nine have been found to be stable on current treatment while three have died." In April 2013, it was reported that of the original 12 patients six had died. The other six responded to medication. Again in September 2013, a teenager case was reported from Kokilaben Hospital in Andheri.

In all these cases, WHO has denied naming these strains as TDR but have embraced the term XXDR or XDR plus. Extensively drug-resistant TB (XDR- TB) is a type of multidrug-resistant tuberculosis  that is resistant to isoniazid and rifampin, plus any fluoroquinolone and at least one of three injectable second-line drugs. In contrast, XXDR TB or XDR Plus TB refers to a strain, which is resistant to all the first and second line Anti-TB drugs. Naming the strains as TDR implies that the bacilli are resistant to everything. In reality, treatment has been achieved in some cases at least with unconventional regimens leading to the idea that there is some left over sensitivity. There are several other reasons why this term is inappropriate. Raviglione etal has written an extensive article arguing the use of the term.

The development of XXDR status in every case has been traced to improper regimen and use of antibiotics. Current recommendations include that both first and second line drugs be tested in the laboratory and then used for treatment.
Migliori GB, De Iaco G, Besozzi G, Centis R, & Cirillo DM (2007). First tuberculosis cases in Italy resistant to all tested drugs. Euro surveillance, 12 (5) PMID: 17868596

Velayati AA, Masjedi MR, Farnia P, Tabarsi P, Ghanavi J, Ziazarifi AH, & Hoffner SE (2009). Emergence of new forms of totally drug-resistant tuberculosis bacilli: super extensively drug-resistant tuberculosis or totally drug-resistant strains in Iran. Chest, 136 (2), 420-5 PMID: 19349380

Udwadia, Z., Amale, R., Ajbani, K., & Rodrigues, C. (2011). Totally Drug-Resistant Tuberculosis in India Clinical Infectious Diseases, 54 (4), 579-581 DOI: 10.1093/cid/cir889

Cegielski P, Nunn P, Kurbatova EV, Weyer K, Dalton TL, Wares DF, Iademarco MF, Castro KG, & Raviglione M (2012). Challenges and controversies in defining totally drug-resistant tuberculosis. Emerging infectious diseases, 18 (11) PMID: 23092736

Friday, June 03, 2016

First case of MCR-1 in US

Table 1: Antibiotic resistance profile.

I have previously posted on Emergence of MCR-1 and how it's existence has been shown in multiple isolates in many different countries. The findings are hot in the international press. See my earlier posts 1, 2, 3) . According to the most recent report, an E coli carrying this gene was isolated from a urine sample from a 49 year old woman from Pennsylvania with no recent travel outside of the United States.

According to the report, the strain was an ESBL producing E coli (ST457 clone). The strain was qPCR positive for MCR-1 gene. In total, the bacterium contained 15 antibiotic-resistance genes on two plasmids. One of them plasmid was a novel IncF plasmid. It was also reported that the strain (Isolate ID- MRSN 388634) and had no genes for carbapenemases. The antibiotic resistance profile is shown in Table 1.

I wouldn't have bothered writing this piece since MCR-1 is now an old news and many countries are reporting this. This is just one case. But the media hype is too much and quite misleading. The reports all over the internet imply that this strain is totally resistant, with a fancy term- Superbug. Yes, this bug is colistin resistant, but not untreatable. As you can see from the antibiogram profile, the strain is not a CRE (Carbapenemase Resistant Enterobacteriaceae). In fact, the MIC value for Ertapenem and Meropenem is <0.25 ug/ml.

Table 2: Characteristics of plasmids in E.coli MRSN 388634.
Patrick McGann, the lead author of the paper comments, "Colistin is one of the last efficacious antibiotics for the treatment of highly resistant bacteria. The emergence of a transferable gene that confers resistance to this vital antibiotic is extremely disturbing. The discovery of this gene in the U.S. is equally concerning and continued surveillance to identify reservoirs of this gene within the military healthcare community and beyond is critical to prevent its spread".

Fig 1: Google hits for keywords- "MCR-1 superbug in US"
Does colistin resistance spell the end? Just look at the google hits for "MCR-1 superbug in US". The headings look scary. I have my opinions and debates about if the headlines really mean what it implies. For example, Proteus mirabilis has an inherent resistance to colistin and multiple other antibiotics. Does that mean it can't be treated? The answer is it can definitely be treated with alternative antibiotics. There are several possible antibiotic options available for a given pathogen. In this case, the isolate was not a CRE and hence carbapenems are the option. I agree that colistin resistance is a concern, but calling the end is probably debatable. 

That brings me to a question. Will there ever be an isolate that is totally drug resistant? If you wish to give an example of TDR-TB then am sorry you are wrong. As per WHO and global consensus, Total Drug Resistance (TDR) is a misnomer since these strains are still susceptible to some antibiotics. As far as my knowledge exists (I maybe ignorant) there never has been a TDR resistance for any organism. There is no reason why this cannot happen in future. But such a probability is less. To be resistance to a huge variety of drugs the strain needs to carry a huge number of genes for it. In the case of E.coli MRSN 388634 (see Table 2), resistance is encoded in 2 plasmids for a huge set of genes. Carrying and managing so many genes is a heavy energetic toll on the organism and given a choice they will drop it. I would contest that, in this case probably carbapenemase resistance was dropped.

At this time surveillance is on to find the possible source. As of April 2016, more than 44,000 Salmonella and 9,000 E coli/ Shigella isolates had their whole genome sequenced without any positive match for MCR-1. But more recently by May 26, 2016, colistin-resistant E. coli has been demonstrated in a single sample from a pig intestine This data comes from testing 949 animal samples screened so far.

Quoting from the HHS blog post, "The two detections of the mcr-1 gene in the U.S. provide a new clue into the antibiotic resistance landscape, and it also highlights how much we still do not understand". As of the case we are discussing with the infection, the women is doing fine. Dr. Beth Bell from CDC comments, "The patient herself is, ya know, fine. But we don't have information yet which can tell us for sure that the infection has been eradicated or not. This bacteria can be spread from human to human by close personal contact, by what we sometimes call the fecal to oral route possibly where there is contamination of food that may not be completely cooked."
McGann P, Snesrud E, Maybank R, Corey B, Ong AC, Clifford R, Hinkle M, Whitman T, Lesho E, & Schaecher KE (2016). Escherichia coli Harboring mcr-1 and blaCTX-M on a Novel IncF Plasmid: First report of mcr-1 in the USA. Antimicrobial agents and chemotherapy PMID: 27230792

Thursday, June 02, 2016

Lab Series# 11- VOPBA


The most important part of any viral life cycle is to get inside a cell. When any new virus is discovered, the most important research that needs to be immediately done is find the receptor in the cell. More easily said than done, I recently came to an understanding that there are some criterions to call a virus receptor as a functional receptor. I have only known part of this story and so I scanned up literature in depth and had some understanding of the concept.

There is no single methodology for identifying virus receptor since the virus can bind to proteins glycans etc. Sometimes a virus can have more than a single receptor or sometimes receptors or viral proteins need to be modified by an enzyme and so on and so forth. However, most of the virus have a straightforward binding with cellular membrane receptors. This where a technique called as Virus Overlay Protein Binding Assay (VOPBA) comes in.

Fig 1: Procedure of VOPBA. Source
The idea and popularisation of VOPBA technique largely credited to Richard Hegele, In an attempt to find the receptor for RSV. In the first experiment, the cellular membrane was digested with enzymes such that you end up with only proteins, fat or carbohydrates. This preparation was then infected with RSV and found that cells lacking protein in cell membrane showed no infection, mean to say that the virus was using proteins as the receptor. The next step is to identify the protein.  For this, the membrane proteins are separated in a gel, blotted out and virus particle is added. The virus binds to the protein, that forms the receptor and is identified by using antibodies against the virus. The gel band is cut, digested and the protein is isolated and identified using Mass Spectrometry. This technique can be used to identify almost any receptor, though the details of the technique varies widely. The technique can be modified by including the receptor array on a microchip (for example glycan array for Influenza), which can be used for identifying the receptor.

One of the most common questions about identification of virus receptor is verification. How do you actually prove that indeed the one identified is the functional receptor? There are a set of criteria. I'm not sure if one should call it postulates of receptor identification, but I have seen literature that implies they are. 
  • Cells lacking the receptor should be resistant to infection.
  • Introducing the receptor molecule in a non-susceptible cell line should / could confer susceptibility.
  • Antibodies against the receptor moiety should/could block infection
  • Competitive binding of the receptor should reduce infection.
The rules look quite straightforward. It should be possible for the virus to infect cells carrying the receptor (That's the whole point). If you are to introduce the receptor into a cell that doesn't have one, that should be infectable too. This may not be true always since some cell lines can be non-permissive. So a better approach is to knock out the protein gene and (or) silence the protein production using siRNA and show reduced infection. Antibodies that bind to the receptor masking the binding site should be able to block infectivity and if there is a competitor protein or chemical that can bind to the receptor it should be possible to reduce the infection rate. I understand that this rule is not universal and cases of exception exist. If at least a couple of rules hold up, then it is safe to call the receptor as a functional receptor.
Bass DM, & Greenberg HB (1992). Strategies for the identification of icosahedral virus receptors. The Journal of clinical investigation, 89 (1), 3-9 PMID: 1309536

Kun-Tong Jia, Chang-Jun Guo, Xiao-Bo Yang, Jian-Guo He. Virus Overlay Assay (Far-Western Blotting). BioProtocols.