List of things to carry while travelling to Andaman and Nicobar Islands

 1.       Cash (Indian currency) as per requirement needs to be carried as the network is poor in certain places and Google pay or any other mode of online payment might not work.

2.       Driver’s license is required if you want to rent a self-driven two-wheeler. On the smaller islands, these are preferred mode of transport.

3.       Comfortable cotton clothes are preferred due to the humid weather. You will also end up visiting many beaches. So knee length dresses would be ideal. Wearing jeans may be uncomfortable and will get filled up with beach sand.

4.       Footwear also needs to be suitable for walking on sand. Shoes are not good for these. Floaters or flat slippers would be fine.

5.       Mosquito repellent can be carried if you are very sensitive to mosquito bites. Generally, this is not a concern.

6.       For pandemic times: You will either need a Covid testing report (within 48 hours prior to arrival) showing negative status needs to be taken with you from the mainland or will need to done upon arrival at the Port Blair airport. The cost of the RAT (Rapid Antigen Test) being done at the Veer Savarkar Airport is 500 INR.

7.       Extra clothing and large towels would be very useful while visiting the beach areas. Numerous water sports in addition to swimming and bathing in the ocean are possible.

Most of the things can be purchased at Port Blair. A good option is the Mubarak Hyper market located at Biggie Line, Port Blair, 744101 (Remember the Pin Code as this is asked in some forms that need to be filled upon arrival). 

A gene seems to be lost in chicken - is it really true?

Plg-RKT or Plasminogen Receptor K terminal was first identified a decade ago (in the year 2010) based on a proteomics study. Given its recent discovery, the number of research papers focused solely on this protein are limited. List of the papers dealing with the discovery and functional characterization of this protein as well as its relevance to understanding the healthy and disease state of the body are provided in the references. Prior to its characterization, Plg-RKT was known as C9orf46 due to its presence on human chromosome 9 (ORF 46). The original discovery of the role of Plg-RKT and most of the subsequent work related to this protein emanate from the lab of Lindsey Miles (Professor of Cell and Molecular Biology) at The Scripps Research Institute, La Jolla, CA. The 2010 paper notes

“Our isolation of peptides corresponding to C9orf46 homolog is, to our knowledge, the first demonstration of the existence of this protein. We have designated the protein, Plg-R_KT, to indicate a plasminogen receptor with a C-terminal lysine and having a transmembrane domain.”

Presence of the C-terminal lysine in this protein seems to be highly conserved across mammals and birds. This lysine residue is exposed on the cell surface and is recognized by plasminogen. The known functions of the Plg-RKT gene can be summarized as follows:

1. Regulation of macrophage phenotype
2. Mammary development and lactation
3. Regulation of efferocytosis
4. Metabolic homeostasis and adipose function
5. Mediation of Lipoprotein(a) endocytosis
6. Regulation of cell surface plasminogen activation

Given the evidence for multiple important functional roles of this gene, it seems unlikely that this gene is dispensable. Several other receptors for plasminogen do exist and could potentially play a compensatory role. The orthologs of Plg-RKT are well conserved and a 1 to 1 ortholog (CG13404 (FBgn0030559)) is annotated in Drosophila melanogaster. A recent pre-print implicates this orthologous gene in Coxiella burnetii Infection susceptibility in Drosophila melanogaster based on a GWAS study that relies on DGRP. Although no ortholog is annotated in yeast, two homologs (tag-280 (WBGene00044322) and tag-281 (WBGene00044323)) are annotated in C. elegans and remain uncharacterised.

To further identify potential gene losses in chicken, we obtained a list of genes that are co-expressed with Plg-RKT in human samples or otherwise known to interact with Plg-RKT and evaluated whether their orthologs are present in chicken. 

Sl. No	Human Gene stable ID	Gene name	Chicken ortholog	Remark
1	ENSG00000062038	CDH3	ENSGALG00000051984	Pseudogene annotation on ensemble but annotated mRNA with ORF at KY120273.1
2	ENSG00000137975	CLCA2	ENSGALG00000050155	Ortholog found
3	ENSG00000149547	EI24	ENSGALG00000038097	Ortholog found
4	ENSG00000126749	EMG1	ENSGALG00000014568	Ortholog found
5	ENSG00000068438	FTSJ1	Is this lost ??	Chicken Chr 12 and Chr 13 breakpoint
6	ENSG00000189280	GJB5	ENSGALG00000054289	Ortholog found
7	ENSG00000108010	GLRX3	ENSGALG00000010464	Ortholog found
8	ENSG00000196743	GM2A	ENSGALG00000027534	Ortholog found
9	ENSG00000138271	GPR87	ENSGALG00000010377	Ortholog found
10	ENSG00000113161	HMGCR	ENSGALG00000014948	Ortholog found
11	ENSG00000053747	LAMA3	ENSGALG00000015056	Ortholog found
12	ENSG00000172172	MRPL13	ENSGALG00000041863	Ortholog found
13	ENSG00000131467	PSME3	ENSGALG00000002937	Ortholog found
14	ENSG00000087494	PTHLH	ENSGALG00000017295	Ortholog found
15	ENSG00000176225	RTTN	ENSGALG00000013745	Ortholog found
16	ENSG00000104549	SQLE	ENSGALG00000036915	Ortholog found
17	ENSG00000056972	TRAF3IP2	ENSGALG00000015026	orthology not annotated
18	ENSG00000087245	MMP2	ENSGALG00000003580	Ortholog found
19	ENSG00000100985	MMP9	ENSGALG00000006992	Ortholog found

Most of the above genes have clear 1 to 1 orthologs in chicken. The origin and diversification of the plasminogen activation system has been explored by looking at homologs of 15 genes consisting of the following groups:

• PLG, HGF and MST-1
• HABP2, HGFAC, tPA and uPA
• SERPINE1 (PAI-1), SERPINE2, SERPINE3 and SERPINI1
• PAI-2
• VTN
• 3LU and uPAR

When the orthologs of these genes are searched in chicken, we again find most of them. The exceptions are PLAUR and SERPINE1. Prior work has suggested these genes are lost in chicken. In addition, to these loss events, we see duplication of PLAU and PLG like loci. The potential loss of PLAUR could be interesting as PLAUR is known to interact with PLG. 

Gene stable ID	Gene name	Chicken gene stable ID
ENSG00000173531	MST1	ENSGALG00000002722
ENSG00000122861	PLAU *	ENSGALG00000050317
ENSG00000122861	PLAU *	ENSGALG00000046993
ENSG00000148702	HABP2	ENSGALG00000008905
ENSG00000163536	SERPINI1	ENSGALG00000009470
ENSG00000122194	PLG *	ENSGALG00000028886
ENSG00000122194	PLG *	ENSGALG00000004293
ENSG00000135919	SERPINE2	ENSGALG00000005135
ENSG00000011422	PLAUR
ENSG00000104368	PLAT	ENSGALG00000003709
ENSG00000253309	SERPINE3	ENSGALG00000017017
ENSG00000109758	HGFAC	ENSGALG00000015623
ENSG00000019991	HGF	ENSGALG00000033974
ENSG00000106366	SERPINE1
ENSG00000109072	VTN	ENSGALG00000003589

We next compiled the list of all the plasminogen receptors from previous reviews.

Sl No	Gene stable ID	Gene symbol	Chicken gene stable ID	Gene name	Remarks
1	ENSG00000074800	ENO1	ENSGALG00000002377	alpha-enolase	First plasminogen receptor to be identified. See: Activation of plasminogen into plasmin at the surface of endothelial microparticles: a mechanism that modulates angiogenic properties of endothelial progenitor cells in vitro (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2495018/)
2	ENSG00000189403	HMGB1	ENSGALG00000042875	Amphoterin
3	ENSG00000197747	S100A10	ENSGALG00000028774	P11
4	ENSG00000107020	PLGRKT	Is this really lost?	Plg-RKT
5	ENSG00000182718	ANXA2	ENSGALG00000003770	Annexin A2
6	ENSG00000170421	KRT8	ENSGALG00000050400	Cytokeratin 8	Orthology not annotated
7	ENSG00000005961	ITGA2B	ENSGALG00000054766	Integrin Alpha-IIb/beta-3
8	ENSG00000138448	ITGAV	ENSGALG00000002655	Integrin AlphaVbeta3
9	ENSG00000169896	ITGAM	Orthologs found in lizard and alligator but not in birds. Duplication of ITGAX (see: Structural analysis of the CD11b gene and phylogenetic analysis of the alpha-integrin gene family demonstrate remarkable conservation of genomic organization and suggest early diversification during evolution.(https://www.jimmunol.org/content/150/2/480.long))	Integrin Subunit Alpha M	Integrin αMβ2 Orchestrates and Accelerates Plasminogen Activation and Fibrinolysis by Neutrophils (https://www.jbc.org/content/279/17/18063.long)
10	ENSG00000160255	ITGB2	ENSGALG00000007511	Integrin subunit beta 2
11	Histone genes occur in cluster and all copies retain high levels of sequence similarity. See: Molecular Evolution of the Nontandemly Repeated Genes of the Histone 3 Multigene Family (https://academic.oup.com/mbe/article/19/1/68/1066713)			Histone 2B	Phosphatidylserine as an anchor for plasminogen and its plasminogen receptor, Histone H2B, to the macrophage surface (https://onlinelibrary.wiley.com/doi/full/10.1111/j.1538-7836.2010.04132.x)

The Immunogenetics journal has previously (2019 [Convergent inactivation of the skin-specific C-C motif chemokine ligand 27 in mammalian evolution (https://link.springer.com/article/10.1007/s00251-019-01114-z)] and 2018 [Cetacea Are Natural Knockouts for IL20 (https://pubmed.ncbi.nlm.nih.gov/29998404/)]) published gene loss stories in cetaceans. However, both IL20 and CCL27 are well studied genes and the observed loss spanned several species including independent losses. The authors could also provide a fairly convincing explanation for why these genes were lost in cetacean species and provide evidence from re-sequencing datasets and RNA-seq experiments. Loss of NLRC4 and NAIP in pigs was reported [Pig lacks functional NLRC4 and NAIP genes (https://link.springer.com/article/10.1007/s00251-016-0955-5)] in 2017. Being a domesticated species, changes in the immune repertoire of the pig has implications for the pork industry. Interestingly, this paper makes a reference to the lack of RIG-I in chicken and how the immune response is different because of this. 

Given all this background information we wanted to make sure we provided enough evidence for the pseudogenisation of PLGRKT to convince the reviewers. The recently published online PseudoChecker tool failed to find the remnants of the PLGRKT gene. Neither was it able to find the intact gene in Duck when the human exons and CDS were used as the reference. So PLGRKT can be added to the list of less than 5% genes that PseudoChecker is supposedly unable to find. Fortunately, we have been told that real lossomicists (scientists whose specialty is finding gene loss events) use exon by exon tblastx followed by careful scrutiny to prove gene loss. So we did this and find very clear evidence for the existence of exon-3 remains and largely intact exon-4 in chicken. Side by side comparison with results with duck cDNA are provided here: https://github.com/ceglab/PLGRKT/tree/master/tblastx. So all this and the evidence presented by Sharma et. al., suggests this genes is truly lost in chicken. 

In addition to the work done by the Miles lab, recently published papers are from the University of Otago (see talk describing the work here (PLGRKT starts around 30 minutes into the video): https://www.youtube.com/watch?v=0bpNZSZdeQU) and Medical University of Vienna (see video here: https://www.youtube.com/watch?v=xjPmTDkhWr8).

 References

1.      Plasminogen and the Plasminogen Receptor, Plg-RKT, Regulate Macrophage Phenotypic, and Functional Changes (https://www.frontiersin.org/articles/10.3389/fimmu.2019.01458/full)

2.      The Plasminogen Receptor, Plg-RKT, and Macrophage Function (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3484331/)

3.      The Plasminogen Receptor, Plg-RKT, is Essential for Mammary Lobuloalveolar Development and Lactation (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5965281/)

4.      The Novel Plasminogen Receptor, Plasminogen ReceptorKT (Plg-RKT), Regulates Catecholamine Release (https://www.jbc.org/content/286/38/33125.full)

5.      Plasminogen receptors and their role in the pathogenesis of inflammatory, autoimmune and malignant disease (https://onlinelibrary.wiley.com/doi/pdf/10.1111/jth.12064)

6.      Deficiency of Plasminogen Receptor, Plg-RKT, Causes Defects in Plasminogen Binding and Inflammatory Macrophage Recruitment in vivo (https://pubmed.ncbi.nlm.nih.gov/27714956/)

7.      Plasminogen and the Plasminogen receptor, Plg-RKT, regulate efferocytosis and macrophage reprogramming (https://www.fasebj.org/doi/abs/10.1096/fasebj.2018.32.1_supplement.280.4)

8.      The Plasminogen Receptor, Plg-RKT, Regulates Metabolic Homeostasis and Promotes Healthy Adipose Function (https://www.ahajournals.org/doi/abs/10.1161/circ.134.suppl_1.19088)

9.      Proteomics-based discovery of a novel, structurally unique, and developmentally regulated plasminogen receptor, Plg-RKT, a major regulator of cell surface plasminogen activation (https://ashpublications.org/blood/article/115/7/1319/26700/Proteomics-based-discovery-of-a-novel-structurally)

10.  Regulation of Macrophage Migration by a Novel Plasminogen Receptor Plg-R KT (https://pubmed.ncbi.nlm.nih.gov/21940822/)

11.  New Insights Into the Role of Plg-RKT in Macrophage Recruitment (https://pubmed.ncbi.nlm.nih.gov/24529725/)

12.  Plasminogen Receptors: The First Quarter Century (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3938387/)

13.  New Insight on the Role of Plasminogen Receptor in Cancer Progression (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4521684/)

14.  Plasminogen Receptors in Human Malignancies: Effects on Prognosis and Feasibility as Targets for Drug Development (https://pubmed.ncbi.nlm.nih.gov/31755385/)

15.  Differential expression of Plg-RKT and its effects on migration of proinflammatory monocyte and macrophage subsets (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6688429/)

16.  Recycling of Apolipoprotein(a) After PlgRKT-Mediated Endocytosis of Lipoprotein(a) (https://www.ahajournals.org/doi/full/10.1161/circresaha.116.310272)

17.  Natural Genetic Variation in Drosophila melanogaster Reveals Genes Associated with Coxiella burnetii Infection  (https://www.biorxiv.org/content/10.1101/2020.05.21.109371v1.full)

18. Origin and diversification of the plasminogen activation system among chordates (https://bmcevolbiol.biomedcentral.com/articles/10.1186/s12862-019-1353-z)

 

Genome sequencing and assembly of Mesua Ferrea or Nagakeshara

The first draft genome assembly of the plant Mesua Ferrea also colloquially known as Nagakeshara has been published by Patil et.al., 2020. Using high coverage (~180X ) Illumina sequencing data, the draft genome has been assembled using the latest genome assembly software. Due to its importance in traditional medicine and use as a biofuel, the plant has also acquired important religious significance. Infact, it has been made the state flower of the North-eastern states of Tripura and Mizoram. The de novo assembly generated by Patil et.al., 2020 is 614 Mega-base pair (Mbp) in size and has an N50 of 392 Kilo-base pairs (Kbp). The assembly quality is thought to be comparable to other published Malpighiales genomes. 

Some genome assemblies aspire to have even higher N50 values to be considered of high quality. To achieve these exceedingly high contiguity values these projects tend to rely upon Pacbio sequencing data or Nanopore sequencing data. In addition to this, some projects utilize optical mapping data also. However, these advanced methods of sequencing are expensive and equipment for these methods are hard to find. However, the manuscript published by Patil et.al., 2020 adds an additional dimension to their study by performing a comparative analysis of the demographic histories of several forest plants using the PSMC program. Notably, the parameter settings used to run PSMC are noted and proper optimization is performed. This is in contrast a slew of papers which tend to ignore the parameter settings that are to be used.

A previous version of the manuscript titled "CoalQC - Quality control while inferring demographic histories from genomic data: Application to forest tree genomes" dealing with various technical aspects now continues to languish on the Biorxiv repository. This may be a good testament to the fact that good English writing skills and proper structuring of the manuscript is more important than technical correctness when publishing in higher impact factor journals. Appeals to the contrary are interesting but unlikely to make much of an impact.

 

Gene Loss Association Study (GLAS) in budding yeast gets published in PeerJ

A central goal in evolutionary biology is the identification of changes in the genome that result in  changes at the level of the phenotype. Associating gene loss events with a specific phenotype has seen increasing interest with the availability of high quality genome assemblies for large numbers of species. A detailed discussion of the approach and examples of associations that have been successfully established is provided in Shinde et al. 2019. Graphical summary of the idea of Gene Loss Association Studies or GLAS is provided in below figure.

Figure 1 from Shinde et al. 2019

Two years ago, while screening the budding yeast genomes to identify associations between gene loss and kinetochore phenotypes, it became apparent that four genes from the inner kinetochore were missing in Naumovozyma species. The Kobayashi et al. 2015 paper had studied centromeres in both Naumovozyma species with genome assemblies using ChIP-seq and established the uncoventional point centromeres. Hence, this association between gene loss and transition to evolutionary neo-centromeres (ENC's) seemed an important observation to better understand the processes involved in generating ENC's. While the manuscript was under preparation, an influential study (Kasinathan et al 2018) about defining features of centromeres was published from the Henikoff lab. This study was important as they compared sequence characteristics of Naumovozyma centromeres in addition to several other species. Continuing on the work from Henikoff lab we compared the dyad density of the old and new centromeres in both Naumovozyma species. 

Being a bird coloration enthusiast i studied plumage coloration in crows during my PhD. A prominent discovery in the field of bird morphs around this time was the identification of a large chromosomal inversion in Ruff's that associates with a morph polymorphism (see Kupper et al 2016). One breakpoint of this inversion is known to disrupt the essential CENP-N gene. As a result of this lethality, individuals homozygous for the inversion allele don't exist. The existence of this balanced polymorphism has been the focus of subsequent research. One of the four genes that were found to be lost in Naumovozyma was CHL4, an ortholog of CENP-N. This is an interesting example of a gene which can be essential in certain species while becoming dispensable in a different group of species. 

Given the recent interest in neo-centromere formation and its evolutionary emergence and role in karyotype evolution, the time seems ripe for understanding the sequence of genetic events involved in such transitions. Role of centromere repositioning in reproductive isolation has also received attention. My manuscript describing the GLAS in budding yeast was submitted to PeerJ for Peer review sometime after the nationwide lockdown took hold of India (18th of June). Manuscript was assessed by 3 reviewers, each of whom gave constructive and detailed comments. Quality of the manuscript definitely improved based on the feedback from these anonymous reviewers and the editor. Since, PeerJ provides an option to publish the reviewer comments along with the manuscript, these comments can be seen online. Revision is due in approximately 40 days with some flexibility as no fixed deadline is provided. This is a very useful feature as the time taken to revise a manuscript can vary widely from one manuscript to another. Overall turnaround time for the reviews seemed reasonable and matched with how detailed the reviewer comments were. First set of reviewer comments were received (on 21st July) approximately 1 month after submission. It took me another one month (19th August) to revise the manuscript as other manuscripts had shorter timelines. PeerJ staff checked the manuscript for various issues to ensure it is properly formatted prior to the second round of review. All this was completed by 26th August. The article was finally accepted on 11th September after a second round of review. PeerJ surprisingly does not have an interactive review system and might be for the better. The total time from submission to acceptance was 86 days (~3 months). Proofing of the article was a very smooth experience that involved annotating the pdf proof and uploading it back again. Shortly after the proof was submitted, the article was scheduled for publication on Sep 29th, 2020. This follows in the tradition of PeerJ publishing articles on every Tuesday and Thursday. The publication is now available on the PeerJ website with the title "Loss of inner kinetochore genes is associated with the transition to an unconventional point centromere in budding yeast"

The appalachian conundrum

We are not living through unprecedented times. Yet, like any other generation we sure do feel so. The ongoing covid-19 epidemic, death of George Floyd, earthquakes and locusts might seem to make the times unprecedented. Let us be assured, the human race has seen much worse and survived to become better. The Spanish flu and Slavery are both thankfully in the past. So one can hope that the pandemic and racism will look like distant past at some point. Unfortunately, now it is not that time yet.

Discrimination against Appalachian people is thought to be real enough to have resulted in the enactment of laws in Cincinnati. Just to clarify, the term Appalachian is not applied to native Americans these days, but rather to Ulster-Scot migrants to the US that have settled in the Appalachian region. Vast majority of these people are white and the potentially derogatory term "redneck" is also used to describe this group which remains mired in poverty. This brings me to the Appalachian Conundrum. It is stated as follows:

Imagine a world, far far away if you will. A tiny planet orbiting a giant start much like our earth and sun. Let us call this planet parth. Parth unlike earth is inhabited by many primate like species that look extremely similar, can all communicate in a common language and have the ability to think. The Conundrum is how would these groups on Parth behave?

Will some species conquer the world and subjugate the others? Would there be a war for resources or dominance? Is it possible for only one species to survive through the strife? Can these species co-exist and cooperate to build a better world? How would this co-existence work? Which groups would make up a greater share of the population on parth? Would there be socio-economic disparities between groups?

These are tough questions to answer. May be an episode of start trek will solve this conundrum for us. 

Role of Hypoxia in breast cancer - Alternative Splicing and Methylation

Missing out on a great opportunity can be suffocating. Suffocation or asphyxiation is the deficiency of oxygen supply in the body. Such deprivation of oxygen is known as hypoxia. Hence, it is probably not surprising that looking for opportunities in hypoxia research can result in such outcomes. The recent Nobel Prize in Physiology was awarded to Semenza and colleagues for their pioneering work on hypoxia. No doubt these scientists have avoided suffocation for so long by incorporating some of the defense mechanisms that the body has developed to counteract the lack of oxygen. The role of hypoxia in tumour microenvironment makes it even more pertinent to understand hypoxia and how it is regulated.

One has to realize that while the body as a whole can experience hypoxia, it is the individual cells that respond to this condition. While some cells might die due to the acute lack of oxygen, certain adjoining cells might manage to survive as the oxygen deficiency was not as pronounced. Understanding this cell to cell heterogeneity in dealing with the lack of oxygen would be next step in unraveling the tumor micro environment. However, this needs sophisticated equipment like the 10x Chromium System that can generate single cell RNA-seq libraries from a cell suspension. Only when you have the instrument you can generate pilot data required by the grant agency. Not surprisingly, you need the said grant money to buy the instrument in the first place. This is probably what smart people call a catch 22. 

We decided to overcome this by categorizing entire tumor samples as hypoxic or normoxic as single cell resolution continues to evade our simple minds. In order to classify the tumor into hypoxic or normoxic we needed a signature that could act as a set of features. The molecular signature database is a great source for obtaining such lists of genes involved in a specific molecular function. These signatures have been meticulously assembled by combing through numerous other published datasets. The hypoxia signature on MSigDb is actually based on approximately 80 other lists taken from various studies (including those published by the great Semenza). In order to evaluate the biological meaningfulness of this signature, we used the ShinyGO tool (as it is very Shiny) to visualize the molecular functions that are prominent in this signature set consisting of 200 genes. 

Hypoxia hallmark signature from MSIGDB enrichment for molecular function (200 genes)

While functions involving sugar metabolism are prominent and make sense, they might not be the ideal set of genes to find hypoxic (single cell like) tumor patients. Hence, we decided to make our own new signature. Details of how this was done is described in the manuscript of Pant et. al., 2020. the new custom signature identified by Pant and colleagues is a leaner list that might help achieve the goal of studying hypoxia heterogeneity by looking at inter-individual variation until intra-individual variation becomes accessible. 

Hypoxia custom signature identified by Pant et.al., 2020

Using this super fantastic new signature that is identified by cleverly combining ChIP-seq and micro-array data, we stratified the public TCGA breast cancer data into hypoxia and normoxic patients. By comparing these two groups of tumors, we hoped to understand the differences that might exists between hypoxic and normoxic regions within the tumor. Since, we are specifically interested in Alternative Splicing (due to its ability to increase complexity without any increase in gene number) and its role in hypoxia, we identified differential spliced isoforms between these two groups. Exonic regions that were isoform specific among these isoforms were identified. Only for these exons, the role of DNA methylation was assessed by looking at correlations between the expression level of these exons and the methylation level of proximal DNA measured using arrays. Code required to replicate the results is available on the github repo here: Hypoxia splicing methylation correlation.