Toward high-resolution population genomics using archaeological samples

The term ‘ancient DNA’ (aDNA) is coming of age, with over 1,200 hits in the PubMed database, beginning in the early 1980s with the studies of ‘molecular paleontology’. Rooted in cloning and limited sequencing of DNA from ancient remains during the pre-PCR era, the field has made incredible progress since the introduction of PCR and next-generation sequencing. Over the last decade, aDNA analysis ushered in a new era in genomics and became the method of choice for reconstructing the history of organisms, their biogeography, and migration routes, with applications in evolutionary biology, population genetics, archaeogenetics, paleoepidemiology, and many other areas. This change was brought by development of new strategies for coping with the challenges in studying aDNA due to damage and fragmentation, scarce samples, significant historical gaps, and limited applicability of population genetics methods. In this review, we describe the state-of-the-art achievements in aDNA studies, with particular focus on human evolution and demographic history. We present the current experimental and theoretical procedures for handling and analysing highly degraded aDNA. We also review the challenges in the rapidly growing field of ancient epigenomics. Advancement of aDNA tools and methods signifies a new era in population genetics and evolutionary medicine research

Population differentiation of Southern Indian male lineages correlates with agricultural expansions predating the caste system

Christina J. Adler, Alan Cooper, Clio S.I. Der Sarkissian and Wolfgang Haak are contributors to the Genographic ConsortiumPrevious studies that pooled Indian populations from a wide variety of geographical locations, have obtained contradictory conclusions about the processes of the establishment of the Varna caste system and its genetic impact on the origins and demographic histories of Indian populations. To further investigate these questions we took advantage that both Y chromosome and caste designation are paternally inherited, and genotyped 1,680 Y chromosomes representing 12 tribal and 19 non-tribal (caste) endogamous populations from the predominantly Dravidian-speaking Tamil Nadu state in the southernmost part of India. Tribes and castes were both characterized by an overwhelming proportion of putatively Indian autochthonous Y-chromosomal haplogroups (H-M69, F-M89, R1a1-M17, L1-M27, R2-M124, and C5-M356; 81% combined) with a shared genetic heritage dating back to the late Pleistocene (10–30 Kya), suggesting that more recent Holocene migrations from western Eurasia contributed, <20% of the male lineages. We found strong evidence for genetic structure, associated primarily with the current mode of subsistence. Coalescence analysis suggested that the social stratification was established 4–6 Kya and there was little admixture during the last 3 Kya, implying a minimal genetic impact of the Varna(caste) system from the historically-documented Brahmin migrations into the area. In contrast, the overall Y-chromosomal patterns, the time depth of population diversifications and the period of differentiation were best explained by the emergence of agricultural technology in South Asia. These results highlight the utility of detailed local genetic studies within India, without prior assumptions about the importance of Varna rank status for population grouping, to obtain new insights into the relative influences of past demographic events for the population structure of the whole of modern India.GaneshPrasad ArunKumar, David F. Soria-Hernanz, Valampuri John Kavitha, Varatharajan Santhakumari Arun, Adhikarla Syama, Kumaran Samy Ashokan, Kavandanpatti Thangaraj Gandhirajan, Koothapuli Vijayakumar, Muthuswamy Narayanan, Mariakuttikan Jayalakshmi, Janet S. Ziegle, Ajay K. Royyuru, Laxmi Parida, R. Spencer Wells, Colin Renfrew, Theodore G. Schurr, Chris Tyler Smith, Daniel E. Platt, Ramasamy Pitchappan, The Genographic Consortiu

Phages from Ganges River curtail in vitro biofilms and planktonic growth of drug resistant Klebsiella pneumoniae in a zebrafish infection model

Abstract Bacteriophages are a promising alternative for curtailing infections caused by multi drug resistant (MDR) bacteria. The objective of the present study is to evaluate phage populations from water bodies to inhibit planktonic and biofilm mode of growth of drug resistant Klebsiella pneumoniae in vitro and curtail planktonic growth in vivo in a zebrafish model. Phage specific to K. pneumoniae (MTCC 432) was isolated from Ganges river (designated as KpG). One-step growth curve, in vitro time kill curve study and in vivo infection model were performed to evaluate the efficacy of phage against planktonic growth. Crystal violet assay and colony biofilm assay was done to determine the action of phages on biofilms. KpG phages had a greater burst size, better bactericidal potential and enhanced inhibitory effect against biofilms formed at liquid air and solid air interfaces. In vivo injection of KpG phages revealed that it did not pose any toxicity to zebrafish as evidenced by liver/brain enzyme profiles and by histopathological analysis. In vitro time kill assay showed a 3 log decline and a 6 log decline in K. pneumoniae colony counts, when phages were administered individually and in combination with streptomycin, respectively. The muscle tissue of zebrafish, infected with K. pneumoniae and treated with KpG phages showed a significant 2 log decline in bacterial counts relative to untreated control. Our study reveals that KpG phages has the potential to curtail plantonic and biofilm mode of growth in vivo in higher animal models.</jats:p

Phages from Ganges River curtail in vitro biofilms and planktonic growth of drug resistant Klebsiella pneumoniae in a zebrafish infection model

AbstractBacteriophages are a promising alternative for curtailing infections caused by multi drug resistant (MDR) bacteria. The objective of the present study is to evaluate phage populations from water bodies to inhibit planktonic and biofilm mode of growth of drug resistant Klebsiella pneumoniae in vitro and curtail planktonic growth in vivo in a zebrafish model. Phage specific to K. pneumoniae (MTCC 432) was isolated from Ganges River (designated as KpG). One-step growth curve, in vitro time kill curve study and in vivo infection model were performed to evaluate the ability of phage to curtail planktonic growth. Crystal violet assay and colony biofilm assay were performed to determine the action of phages on biofilms. KpG phages had a greater burst size, better bactericidal potential and enhanced inhibitory effect against biofilms formed at liquid air and solid air interfaces. In vitro time kill assay showed a 3 log decline and a 6 log decline in K. pneumoniae colony counts, when phages were administered individually and in combination with streptomycin, respectively. In vivo injection of KpG phages revealed that it did not pose any toxicity to zebrafish as evidenced by liver/brain enzyme profiles and by histopathological analysis. The muscle tissue of zebrafish, infected with K. pneumoniae and treated with KpG phages alone and in combination with streptomycin showed a significant 77.7% and 97.2% decline in CFU/ml, respectively, relative to untreated control. Our study reveals that KpG phages has the potential to curtail plantonic and biofilm mode of growth in higher animal models.</jats:p

Phages from Ganges River curtail in vitro biofilms and planktonic growth of drug resistant Klebsiella pneumoniae in a zebrafish infection model

Abstract Bacteriophages are a promising alternative for curtailing infections caused by multi drug resistant (MDR) bacteria. The objective of the present study is to evaluate phage populations from water bodies to inhibit planktonic and biofilm mode of growth of drug resistant Klebsiella pneumoniae in vitro and curtail planktonic growth in vivo in a zebrafish model. Phage specific to K. pneumoniae (MTCC 432) was isolated from Ganges River (designated as KpG). One-step growth curve, in vitro time kill curve study and in vivo infection model were performed to evaluate the ability of phage to curtail planktonic growth. Crystal violet assay and colony biofilm assay were performed to determine the action of phages on biofilms. KpG phages had a greater burst size, better bactericidal potential and enhanced inhibitory effect against biofilms formed at liquid air and solid air interfaces. In vitro time kill assay showed a 3 log decline and a 6 log decline in K. pneumoniae colony counts, when phages were administered individually and in combination with streptomycin, respectively. In vivo injection of KpG phages revealed that it did not pose any toxicity to zebrafish as evidenced by liver/brain enzyme profiles and by histopathological analysis. The muscle tissue of zebrafish, infected with K. pneumoniae and treated with KpG phages alone and in combination with streptomycin showed a significant 77.7% and 97.2 % decline in CFU/ml, respectively, relative to untreated control. Our study reveals that KpG phages has the potential to curtail plantonic and biofilm mode of growth in vivo in higher animal models.</jats:p

Phages from Ganges River curtail in vitro biofilms and planktonic growth of drug resistant Klebsiella pneumoniae in a zebrafish infection model

Abstract Bacteriophages are a promising alternative for curtailing infections caused by multi drug resistant (MDR) bacteria. The objective of the present study is to evaluate phage populations from water bodies to inhibit planktonic and biofilm mode of growth of drug resistant Klebsiella pneumoniae in vitro and curtail planktonic growth in vivo in a zebrafish model. Phage specific to K. pneumoniae (MTCC 432) was isolated from Ganges river (designated as KpG). One-step growth curve, in vitro time kill curve study and in vivo infection model were performed to evaluate the efficacy of phage to curtail planktonic growth. Crystal violet assay and colony biofilm assay was done to determine the action of phages on biofilms. KpG phages had a greater burst size, better bactericidal potential and enhanced inhibitory effect against biofilms formed at liquid air and solid air interfaces. In vivo injection of KpG phages revealed that it did not pose any toxicity to zebrafish as evidenced by liver/brain enzyme profiles and by histopathological analysis. In vitro time kill assay showed a 3 log decline and a 6 log decline in K. pneumoniae colony counts, when phages were administered individually and in combination with streptomycin, respectively. The muscle tissue of zebrafish, infected with K. pneumoniae and treated with KpG phages showed a significant 2 log decline in bacterial counts relative to untreated control. Our study reveals that KpG phages has the potential to curtail plantonic and biofilm mode of growth in vivo in higher animal models.</jats:p

Restoring colistin sensitivity in colistin-resistant E. coli: Combinatorial use of MarR inhibitor with efflux pump inhibitor

AbstractAntibiotics like colistin are the last resort to deal with infections by carbapenem-resistant Enterobacteriaceae (CREB). Resistance to colistin severely restricts therapeutic options. To tackle this dire situation, urgent measures to restore colistin sensitivity are needed. In this study, whole-genome sequencing of colistin-resistant E. coli strain was performed and the genome analysis revealed that the strain belonged to the sequence type ST405. Multiple mutations were observed in genes implicated in colistin resistance, especially those related to the L-Ara-4-N pathway but mgrB was unmutated and mcr1-9 genes were missing. MarR inhibitor salicylate was used to re-sensitize this strain to colistin, which increased the negative charge on the cell surface especially in colistin resistant E. coli (U3790 strain) and thereby facilitated a decrease in colistin MIC by 8 fold. It is indeed well known that MarR inhibition by salicylate triggers the expression of AcrAB efflux pumps through MarA. So, in order to fully restore colistin sensitivity, a potent efflux pump inhibitor (BC1), identified earlier by this group was employed. The combination of colistin with both salicylate and BC1 caused a remarkable 6 log reduction in cell counts of U3790 in time-kill assay. Infection of muscle tissue of zebrafish with U3790 followed by various treatments showed that the combination of colistin + salicylate + BC1 was highly effective in reducing bioburden in infected muscle tissue by 4 log fold. Thus, our study shows that a combination of MarR inhibitor to enhance colistin binding and efflux pump inhibitor to reduce colistin extrusion was highly effective in restoring colistin sensitivity in colistin-resistant clinical isolate of E. coli in vitro and in vivo.</jats:p

Role of Next Generation Sequencing in the Diagnosis of Unexplained Thrombocytopenias- an Experience at a Tertiary Care Hospital

Abstract BACKGROUND Next Generation Sequencing (NGS) has been enormously rewarding in the field of diagnostic hematology. In particular, the diagnosis of inherited disorders has progressed in leaps and bounds. These patients tend to remain undiagnosed for a long period of time not only because of unavailability of molecular diagnostics but also due to lack of cognizance and atypical presentations. Thrombocytopenia (TCP) is a common hematological presentation and can lead to chronic hospital visits to life-threatening bleeds. Most of these patients have acquired disorders such as immune TCP, malignancies, liver disease etc. However, some of them are likely to have unidentified inherited causes. We thus intended to study the utility of NGS in the definitive diagnosis of unexplained TCPs with or without other cytopenias to understand the clinicopathologic characteristics of these patients. METHODOLOGY This was a retrospective descriptive study done at two centres over three years from May 2018 to May 2021. Patients with TCP with one of the following: (a) positive family history (b) clinical/ laboratory clues to an inherited cause (c) chronic TCP with no response to conventional therapies and sent for clinical exome sequencing done by NGS were included in the study. Patients who were negative for germline mutations were excluded. Sequencing of targeted genes was performed on the Illumina platform with a mean coverage of &amp;gt;80-100X. Mutations annotated as pathogenic, likely pathogenic and variant of uncertain significance (VUS) were considered clinically significant. VUS are mutations that are difficult to classify as pathogenic and require clinical validation and family testing. RESULTS Our cohort included 18 patients and were divided into two groups- cases of isolated TCP and cases of TCP with anemia and/or neutropenia. Patients presenting with isolated thrombocytopenia We had nine cases of isolated TCP out of which there were three cases of X-linked macrothrombocytopenia with MYH9 mutation, two cases of Wiskott Aldrich syndrome (WAS) and one case each of congenital thrombotic thrombocytopenic purpura (TTP), atypical Hemolytic Uremic Syndrome (HUS), Fanconi anemia (FA) and grey platelet syndrome. The demographic and mutational characteristics are described in Table 1. Clinical and laboratory clues were present in 7 cases, such as chronic kidney disease, micro/ macrothrombocytopenia, neutrophil inclusions etc. Bone marrow examination was carried out in 4 cases- the significant dyspoiesis in FA and myelofibrosis in grey platelet syndrome mislead to a diagnosis of MDS and myelofibrosis respectively. Seven patients had received treatment with steroids, immunosuppressants, splenectomy, danazol and TPO mimetics before the NGS diagnosis. Patients presenting with thrombocytopenia and other cytopenias This group consisted of nine cases with two cases each of Dyskeratosis Congenita (DKC) and WAS and one case each of TTP, Ghosal hematodiaphyseal dysplasia, Congenital Amegakaryocytic Thrombocytopenia (CAMT), B-cell immunodeficiency with hypogammaglobulinemia type-25 and double homozygous for FA and DKC. The majority of patients in this group were young and had lower platelet counts (Table 2). The most common associated cytopenia was anemia. Phenotypic clues to diagnosis were present in cases of DKC and WAS. The common differentials considered in this group were inherited bone marrow failure syndromes (IBMFS), congenital immunodeficiency syndromes etc. Bone marrow examination was done more frequently in these patients and showed hypocellular marrow in IBMFS and absent megakaryocytes in CAMT. These patients have also been treated with steroids, IVIg, danazol and TPO mimetics. CONCLUSION Patients with inherited isolated TCPs have a chronic course and heterogenous causes therefore tend to be diagnosed later in life. However, patients with TCPs and other cytopenias tend to present at a younger age with infrequent family history. IBMFS was the most common disorder identified in this latter group of patients. Positive family history, clinical and laboratory clues and absence of response to conventional therapies should prompt workup of inherited causes by NGS to avoid long term ineffectual treatment. Further, NGS mutations, in particular VUS have to be interpreted with caution with the help of parental study, clinical presentation, in-silico analysis and inputs from molecular and genetic experts. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare. </jats:sec