Individual Sequences Research Articles

Combinatorial batching of DNA for ultralow-cost detection of pathogenic variants

BackgroundNext-generation sequencing (NGS) based population screening holds great promise for disease prevention and earlier diagnosis, but the costs associated with screening millions of humans remain prohibitive. New methods for population genetic testing that lower the costs of NGS without compromising diagnostic power are needed.MethodsWe developed double batched sequencing where DNA samples are batch-sequenced twice — directly pinpointing individuals with rare variants. We sequenced batches of at-birth blood spot DNA using a commercial 113-gene panel in an explorative (n = 100) and a validation (n = 100) cohort of children who went on to develop pediatric cancers. All results were benchmarked against individual whole genome sequencing data.ResultsWe demonstrated fully replicable detection of cancer-causing germline variants, with positive and negative predictive values of 100% (95% CI, 0.91–1.00 and 95% CI, 0.98–1.00, respectively). Pathogenic and clinically actionable variants were detected in RB1, TP53, BRCA2, APC, and 19 other genes. Analyses of larger batches indicated that our approach is highly scalable, yielding more than 95% cost reduction or less than 3 cents per gene screened for rare disease-causing mutations. We also show that double batched sequencing could cost-effectively prevent childhood cancer deaths through broad genomic testing.ConclusionsOur ultracheap genetic diagnostic method, which uses existing sequencing hardware and standard newborn blood spots, should readily open up opportunities for population-wide risk stratification using genetic screening across many fields of clinical genetics and genomics.

Comparison of post contrast fluid attenuated inversion recovery, 3D T1-SPACE, and T1W MRI sequences with fat suppression in the diagnosis of infectious meningitis.

To assess the usefulness of post contrast Fluid attenuated inversion recovery (FLAIR), 3D T1-SPACE, and T1W magnetic resonance imaging (MRI) sequences with fat suppression in diagnosis of infectious meningitis. 75 patients with clinical suspicion of meningitis were evaluated with post contrast FLAIR (PC-FLAIR), post contrast T1-SPACE (PC-T1-SPACE), and post contrast T1WI (PC-T1WI). Sensitivity, specificity, positive predictive value, and negative predictive value of individual sequences were assessed. The sensitivity of PC-FLAIR (88.4%) was greater than PC-T1-SPACE (85.5%) and PC-T1WI (82.6%), considering cerebrospinal fluid (CSF) analysis as gold standard (p < 0.05). Kappa inter-rater agreement between two radiologists was 0.921 for PC-T1-SPACE, 0.921 for PC-T1WI, and 1.0 for PC-FLAIR with a p value <0.05. Both PC-T1-SPACE and PC-FLAIR performed equally in sulcal space enhancement. PC-T1-SPACE and PC-T1WI performed better in evaluation of pachymeningeal enhancement, ependymal enhancement in cases of ventriculitis, whereas PC-FLAIR was more sensitive in assessment of basal cistern enhancement and enhancement along the cerebellar folia. Meningeal enhancement could be better appreciated in PC-FLAIR image than PC-T1WI and PC-T1-SPACE. Enhancement in PC-T1-SPACE was comparable to that of PC-T1WI. Being a T1 based spin echo sequence, PC-T1-SPACE has all the advantages of PC-T1WI in addition to its ability to differentiate meningeal enhancement from leptomeningeal vessels. Hence, PC-T1WI can be replaced by PC-T1-SPACE and PC-FLAIR can be added to routine MRI protocol in suspected case of meningitis.

High degree of individual repeatability found in the annual migrations of walleye (Sander vitreus) in eastern Lake Ontario

Adult walleye (Sander vitreus) conduct annual migrations from riverine spawning sites in the Bay of Quinte into the eastern basin of Lake Ontario to forage. Although fidelity to spawning river is quite high for these fish across the Laurentian Great Lakes, very little is known about the repeatability of site selection along the entirety of their annual migration. This study used multiple years of acoustic telemetry (2018–2020) to assess the within-individual repeatability of the timing associated with major migratory events (spawning river entry and departure, main lake entry and departure), as well as the average monthly spatial distribution of individuals throughout the year. Fine-scale sequence analysis was used to combine these two metrics to assess the within and among individual sequence dissimilarity in the population. Within-individual repeatability was high across most timing and spatial measures, and the sequence dissimilarity within individual sequences was significantly less than that among individuals. Sex and size were not significantly related to sequence dissimilarity, while individuals from the Trent River displayed more dissimilarity than those from the Napanee River. The high level of within-individual annual repeatability in migrations was demonstrated not only during spawning activity, but at a daily level throughout the entire year. This research shows that walleye possess the ability to orient themselves and navigate across long distances in a highly repeatable manner at all times of the year. Future work will be required to better understand the underlying mechanisms and drivers behind these abilities.

Abstract P4-01-03: Progression-free survival and patient-reported outcomes in HR+, HER2– ABC patients treated with first-line ribociclib + endocrine therapy (ET) or ET monotherapy or chemotherapy in real world setting: 5th interim analysis of RIBANNA

Abstract Background: Ribociclib in combination with endocrine therapy (ET) has demonstrated survival benefits in a broad patient population with hormone receptor–positive (HR+), human epidermal growth factor receptor 2–negative (HER2–) advanced breast cancer (ABC). RIBANNA (CLEE011ADE03) is an ongoing, prospective, noninterventional study assessing the efficacy and safety of first-line (1L) ribociclib in combination with ET in routine clinical practice in women with HR+, HER2– ABC. This study will provide insights into the use of ribociclib in combination with ET with regard to effectiveness and patient-reported outcomes (PRO) in the real-world setting in a large patient population. Methods: Patients with HR+, HER2– ABC starting their 1L treatment with ribociclib + ET, or ET monotherapy, or chemotherapy (CT) were included. Data after disease progression to second-line (2L) and further lines of therapy are being collected too. The progression-free survival (PFS) in 1L, 2L and PFS2 (time from inclusion into the trial in the 1L setting until progression from 2L to third-line therapy) for individual treatment sequences will be analyzed using the Kaplan–Meier method. PRO for all the patients in the 1L and 2L treatment cohorts are being evaluated using the Morisky Medication Adherence Scale (MMAS-8), questionnaires related to quality-of-life (EORTC QLQ-C30) and its breast cancer–specific module (EORTC QLQ-BR23) as well as the Hospital Anxiety and Depression Scale (HADS). Data were collected at baseline and at every 3 months until the end of treatment. Results: By the cutoff date (October 11, 2021) of the fourth interim analysis, data were available for 2187 patients (Table 1). Overall, 633 patients progressed after 1L therapy, including 27.6%, 30.6%, and 43.4% of patients from the 1L ribociclib + ET, ET monotherapy, and CT cohorts, respectively. A total of 286 patients received CDK4/6 inhibitors in 2L, which represents 48.3%, 37.3%, and 27.0% of patients from the 1L ribociclib + ET, ET monotherapy, and CT cohorts, respectively. The PFS in 1L, 2L and PFS2 results for individual treatment sequences will be presented at SABCS 2022. PRO compliance rates at baseline were 88.2%, 89.8%, and 89.4% for EORTC QLQ-C30 global health status, QLQ-BR23, and HADS-D/A, respectively, in the overall population, and 84.5% for MMAS-8 in the 1L ribociclib + ET cohort. Data for patient-reported adherence to 1L ribociclib + ET and for questionnaires related to global health-related quality of life, functioning, and symptoms from patients receiving treatment in 1L and 2L settings will be analyzed and presented at SABCS 2022. Conclusion: The RIBANNA study has shown diverse population characteristics among patients who received ribociclib treatment in a real-world setting. The 5th interim analysis is planned in October 2022. Data on PFS, PFS2, specific therapy sequences and PRO from 1L and 2L that will provide insights on therapy sequencing strategy will be presented at SABCS 2022. Table 1. Patient disposition at the data cutoff date (October 11, 2021). Citation Format: Peter A. Fasching, Cosima Brucker, Thomas Decker, Anne Engel, Thomas Göhler, Christian Jackisch, Jan Janssen, Andreas Köhler, Kerstin Lüdtke-Heckenkamp, Diana Lüftner, Marion van Mackelenbergh, Frederik Marmé, Arnd Nusch, Beate Rautenberg, Toralf Reimer, Marcus Schmidt, Rudolf Weide, Pauline Wimberger, Christian Roos, Achim Wöckel. Progression-free survival and patient-reported outcomes in HR+, HER2– ABC patients treated with first-line ribociclib + endocrine therapy (ET) or ET monotherapy or chemotherapy in real world setting: 5th interim analysis of RIBANNA [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-01-03.

Abstract P4-01-31: High Incidence of Palbociclib Related Neutropenia in Asian Patients Associated with Genetic Polymorphisms

Abstract CDK4/6 inhibitors (CDK4/6i) is well established as the current standard of care for hormone positive (HR(+)) metastatic breast cancer (mBC), in combination with endocrine therapy. CDK4/6i extend a chemotherapy-free, progression free survival (PFS) that preserves quality of life in patients. The most common side effect of CDK4/6i is myelosuppression, with neutropenia the most prevalent adverse effect, especially for palbociclib and ribociclib. In PALOMA-2 and MONALEESA-2, where neutropenia was prevalent (any grade neutropenia 79.5%, grade 3/4 neutropenia 66.5% in PALOMA-2, and any grade neutropenia: 74.3%, grade 3/4 55.3% in MONALEESA-2), although febrile neutropenia seldom occurred (1.8%). Several studies have proposed different genetic factors predisposing to CDK4/6 inhibitor induced neutropenia, including Duffy antigen polymorphisms, ABCB1 and ERCC1 polymorphisms (ABCB1_rs1128503, ABCB1_rs1045642, ERCC1_rs11615, ERCC1_rs3212986), CDK6 polymorphisms, and others. Subgroup studies from the PALOMA trials have suggested that Asian patients receiving palbociclib have higher rates of neutropenia, although the exact explanation is unknown. We conducted a retrospective analysis of 102 Taiwanese patients who received palbociclib for HR(+) mBC at the Taipei Veterans General Hospital for the clinical features, incidence and time course of neutropenia were analyzed. Significant neutropenia incidence was observed (&amp;gt; 95% of all grade neutropenia, 74.5% grade 3/4 neutropenia). In addition, in our clinical cohort, one of the patients was a long term peritoneal dialysis patient newly diagnosed of HR(+) mBC and started on Palbociclib, initially 125mg, later decreased dosage to 100mg after neutropenia occurrence. By Liquid chromatography–mass spectrometry (LC–MS), we quantified the levels of palbociclib in her serum and peritoneal fluid at various time points, while on different dosage use. To our knowledge, this is the first pharmacokinetics analysis on a peritoneal dialysis patient receiving CDK4/6i. To investigate a possible genetic association for the high prevalence of neutropenia, we queried the Taiwan biobank, a nationwide biospecimen repository with 129,586 healthy individual genome wide DNA sequencing data deposited. We investigated the prevalence of the 4 SNPs previously reported to be related to neutropenia in the PALOMA studies. By comparing the Taiwan biobank data to SNP databases from different ethnicities, we observed interesting findings on ethnicity differences of SNP distribution and higher prevalence of neutropenia SNPs. Collectively, in this study we report real world data, biobank genome wide analysis, and a novel report of pharmacokinetic study in a peritoneal dialysis patient, providing novel insights into the real world use of palbociclib and CDKi. Table 1. Median TTF of each subsequent therapy after PAL+ET Characteristics of the clinical cohort treated with palbociclib at TVGH Citation Format: Kuan-Jung Huang, Ting-Hao Kuo, Ta-Chung Chao, Chun-Yu Liu, Yi-Fang Tsai, Chi-Cheng Huang, Ling-Ming Tseng, Cheng-Chih Richard Hsu, Jiun-I Lai. High Incidence of Palbociclib Related Neutropenia in Asian Patients Associated with Genetic Polymorphisms [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-01-31.

Profiling DNA Ligase Substrate Specificity with a Pacific Biosciences Single-Molecule Real-Time Sequencing Assay.

DNA ligases catalyze the joining of breaks in nucleic acid backbones and are essential enzymes for in vivo genome replication and repair across all domains of life. These enzymes are also critically important to in vitro manipulation of DNA in applications such as cloning, sequencing, and molecular diagnostics. DNA ligases generally catalyze the formation of a phosphodiester bond between an adjacent 5'-phosphate and 3'-hydroxyl in DNA, but they exhibit different substrate structure preferences, sequence-dependent biases in reaction kinetics, and variable tolerance for mismatched base pairs. Information on substrate structure and sequence specificity can inform both biological roles and molecular biology applications of these enzymes. Given the high complexity of DNA sequence space, testing DNA ligase substrate specificity on individual nucleic acid sequences in parallel rapidly becomes impractical when a large sequence space is investigated. Here, we describe methods for investigating DNA ligase sequence bias and mismatch discrimination using Pacific Biosciences Single-Molecule Real-Time (PacBio SMRT) sequencing technology. Through its rolling-circle amplification methodology, SMRT sequencing can give multiple reads of the same insert. This feature permits high-quality top- and bottom-strand consensus sequences to be determined while preserving information on top-bottom strand mismatches that can be obfuscated or lost when using other sequencing methods. Thus, PacBio SMRT sequencing is uniquely suited to measuring substrate bias and enzyme fidelity through multiplexing a diverse set of sequences in a single reaction. The protocols describe substrate synthesis, library preparation, and data analysis methods suitable for measuring fidelity and bias of DNA ligases. The methods are easily adapted to different nucleic acid substrate structures and can be used to characterize many enzymes under a variety of reaction conditions and sequence contexts in a rapid and high-throughput manner. © 2023 New England Biolabs and The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of overhang DNA substrates for ligation Basic Protocol 2: Preparation of ligation fidelity libraries Support Protocol 1: Preparation of ligation libraries for PacBio Sequel II sequencing Support Protocol 2: Loading and sequencing of a prepared library on the Sequel II instrument Basic Protocol 3: Computational processing of ligase fidelity sequencing data.

Fitting stochastic epidemic models to gene genealogies using linear noise approximation.

Phylodynamics is a set of population genetics tools that aim at reconstructing demographic history of a population based on molecular sequences of individuals sampled from the population of interest. One important task in phylodynamics is to estimate changes in (effective) population size. When applied to infectious disease sequences such estimation of population size trajectories can provide information about changes in the number of infections. To model changes in the number of infected individuals, current phylodynamic methods use non-parametric approaches (e.g., Bayesian curve-fitting based on change-point models or Gaussian process priors), parametric approaches (e.g., based on differential equations), and stochastic modeling in conjunction with likelihood-free Bayesian methods. The first class of methods yields results that are hard to interpret epidemiologically. The second class of methods provides estimates of important epidemiological parameters, such as infection and removal/recovery rates, but ignores variation in the dynamics of infectious disease spread. The third class of methods is the most advantageous statistically, but relies on computationally intensive particle filtering techniques that limits its applications. We propose a Bayesian model that combines phylodynamic inference and stochastic epidemic models, and achieves computational tractability by using a linear noise approximation (LNA) - a technique that allows us to approximate probability densities of stochastic epidemic model trajectories. LNA opens the door for using modern Markov chain Monte Carlo tools to approximate the joint posterior distribution of the disease transmission parameters and of high dimensional vectors describing unobserved changes in the stochastic epidemic model compartment sizes (e.g., numbers of infectious and susceptible individuals). In a simulation study, we show that our method can successfully recover parameters of stochastic epidemic models. We apply our estimation technique to Ebola genealogies estimated using viral genetic data from the 2014 epidemic in Sierra Leone and Liberia.

ProteInfer, deep neural networks for protein functional inference.

Predicting the function of a protein from its amino acid sequence is a long-standing challenge in bioinformatics. Traditional approaches use sequence alignment to compare a query sequence either to thousands of models of protein families or to large databases of individual protein sequences. Here we introduce ProteInfer, which instead employs deep convolutional neural networks to directly predict a variety of protein functions - Enzyme Commission (EC) numbers and Gene Ontology (GO) terms - directly from an unaligned amino acid sequence. This approach provides precise predictions which complement alignment-based methods, and the computational efficiency of a single neural network permits novel and lightweight software interfaces, which we demonstrate with an in-browser graphical interface for protein function prediction in which all computation is performed on the user's personal computer with no data uploaded to remote servers. Moreover, these models place full-length amino acid sequences into a generalised functional space, facilitating downstream analysis and interpretation. To read the interactive version of this paper, please visit https://google-research.github.io/proteinfer/.

Genetic Diversity of Polymyxin-Resistance Mechanisms in Clinical Isolates of Carbapenem-Resistant Klebsiella pneumoniae: a Multicenter Study in China.

Polymyxin has been the last resort to treat multidrug-resistant Klebsiella pneumonia. However, recent studies have revealed that polymyxin-resistant carbapenem-resistant Klebsiella pneumonia (PR-CRKP) emerged due to the mutations in chromosomal genes or the plasmid-harboring mcr gene, leading to lipopolysaccharide modification or efflux of polymyxin through pumps. Further surveillance was required. In the present study we collected PR-CRKP strains from 8 hospitals in 6 provinces/cities across China to identify the carbapenemase and polymyxin resistance genes and epidemiological features by whole-genome sequencing (WGS). The broth microdilution method (BMD) was performed to determine the MIC of polymyxin. Of 662 nonduplicate CRKP strains, 15.26% (101/662) were defined as PR-CRKP; 10 (9.90%) were confirmed as Klebsiella quasipneumoniae by WGS. The strains were further classified into 21 individual sequence types (STs) by using multilocus sequence typing (MLST), with ST11 being prevalent (68/101, 67.33%). Five carbapenemase types were identified among 92 CR-PRKP, blaKPC-2 (66.67%), blaNDM-1 (16.83%), blaNDM-5 (0.99%), blaIMP-4 (4.95%), and blaIMP-38 (0.99%). Notably, 2 PR-CRKP strains harbored both blaKPC-2 and blaNDM-1. The inactivation of mgrB, associated significantly with high-level polymyxin resistance, was mainly caused by the insertion sequence (IS) insertion (62.96%, 17/27). Furthermore, acrR was inserted coincidently by ISkpn26 (67/101, 66.33%). The deletion or splicing mutations of crrCAB were significantly associated with ST11 and KL47 (capsule locus types), and diverse mutations of the ramR gene were identified. Only one strain carried the mcr gene. In summary, the high IS-inserted mgrB inactivation, the close relationship between ST11 and the deletion or splicing mutations of the crrCAB, and the specific features of PR-K. quasipneumoniae constituted notable features of our PR-CRKP strains in China. IMPORTANCE Polymyxin-resistant CRKP is a serious public health threat whose resistance mechanisms should be under continuous surveillance. Here, we collected 662 nonduplicate CRKP strains across China to identify the carbapenemase and polymyxin resistance genes and epidemiological features. Polymyxin resistance mechanism in 101 PR-CRKP strains in China were also investigated, 9.8% of which (10/101) were K. quasipneumoniae, as determined via WGS, and inactivation of mgrB remained the most crucial polymyxin resistance mechanism, significantly related to high-level resistance. Deletion or splicing mutations of crrCAB were significantly associated with ST11 and KL47. Diverse mutations of the ramR gene were identified. The plasmid complementation experiment and mRNA expression analysis further confirmed that the mgrB promoter and ramR played a critical role in polymyxin resistance. This multicenter study contributed to the understanding of antibiotic resistance forms in China.

Transcriptomic profiling of castes and of sexually and parthenogenetically produced reproductive females in the termite Cavitermes tuberosus

AbstractIn termites with a true worker caste, the development pattern splits up from early developmental stages: primary reproductives develop through the nymphal line, whereas workers and soldiers follow the apterous developmental line. In some species, such as Cavitermes tuberosus Emerson (Isoptera: Termitidae, Termitinae), secondary reproductives (or neotenics) may also develop through the nymphal line from a transitional stage called aspirant. Aspirants originate mostly from automictic parthenogenetic reproduction. Therefore, C. tuberosus queens originate from sexual (primary queens) or parthenogenetic (neotenic queens) reproduction. A comparison of these two queen castes offers the possibility to better understand core molecular underpinnings of caste development and plasticity in termites. We investigated these molecular mechanisms by using high‐throughput Illumina RNA sequencing of pooled individuals. We first assembled the de novo reference transcriptome of C. tuberosus, and then identified the transcripts consistently co‐expressed across castes, sexes, and two alternative routes to female reproduction. Cavitermes tuberosus final transcriptome had 130 874 transcripts, N50 of 3398, and total length of 213 549 184 bp. We found that female reproductive maturation was characterized by gene expression down‐regulation: primary queens expressed fewer transcripts overall and had the greatest number of down‐regulated transcripts when compared to all other castes. In both secondary and primary queens, biological processes involved in muscle development and contraction, flight, and olfactory learning were enriched in the down‐regulated gene cluster. In contrast, processes related to reproductive development, insulin receptor signaling pathway, isoprenoid biosynthesis, and multiple metabolic processes were enriched among up‐regulated genes in primary queens. Finally, we found that 17% of all transcripts (21852) were differently co‐expressed when females from sexual and parthenogenetic origins were compared, even though the expression profile of core reproductive‐related gene clusters showed a similar trend in all reproductive females despite their origin. Our findings fit the genomic imprinting model predictions of a maternal effect that commonly regulates the expression of core reproductive genes in females from parthenogenetic and non‐parthenogenetic origins, whereas the expression of non‐reproductive genes varies.

Screening of novel peptides that specifically interact with vitamin D bound biocomplex proteins

The majority of the vitamin D that is present in the blood binds to vitamin D binding protein (VDBP) and circulates in the form of a complex (VDBP-Complex). Knowing the level of vitamin D in the body is crucial for vitamin D-related treatments so that the right dosage of vitamin D can be given. In other words, it is essential to distinguish between the protein VDBP and the complex form bound to vitamin D. As a novel way for the detection of VDBP-Complex, a more effective phage display methodology was applied in this study along with the addition of two approaches. In order to screen a sequence specific to the target only, the pre-binding method and after-binding method were performed. VDBP-Complex was directly coated on the petri dishes. In order to select phages that specifically bind to the VDBP-Complex, random phages were attached, and selected by 7 times of biopanning. Individual DNA sequences were analyzed for each biopanning to find specific peptide sequences for VDBP-Complex. The affinity of binding phages was verified by ELISA assay using an anti-M13 antibody. The phage having a sequence of SFTKTSTFTWRD (called as M3) has shown the highest binding affinity to VDBP-Complex. As a result of the removal test of VDBP-Complex using magnetic beads conjugated with M3 peptide, it was confirmed that significant decrease of VDBP-Complex. The unique characteristic of the M3 sequence was confirmed through a sequence-modified peptide (SFT motif). That is, it is expected that the M3 peptide may be used to determine the vitamin D levels in the blood.

Ultradeep characterisation of translational sequence determinants refutes rare-codon hypothesis and unveils quadruplet base pairing of initiator tRNA and transcript.

Translation is a key determinant of gene expression and an important biotechnological engineering target. In bacteria, 5'-untranslated region (5'-UTR) and coding sequence (CDS) are well-known mRNA parts controlling translation and thus cellular protein levels. However, the complex interaction of 5'-UTR and CDS has so far only been studied for few sequences leading to non-generalisable and partly contradictory conclusions. Herein, we systematically assess the dynamic translation from over 1.2 million 5'-UTR-CDS pairs in Escherichia coli to investigate their collective effect using a new method for ultradeep sequence-function mapping. This allows us to disentangle and precisely quantify effects of various sequence determinants of translation. We find that 5'-UTR and CDS individually account for 53% and 20% of variance in translation, respectively, and show conclusively that, contrary to a common hypothesis, tRNA abundance does not explain expression changes between CDSs with different synonymous codons. Moreover, the obtained large-scale data provide clear experimental evidence for a base-pairing interaction between initiator tRNA and mRNA beyond the anticodon-codon interaction, an effect that is often masked for individual sequences and therefore inaccessible to low-throughput approaches. Our study highlights the indispensability of ultradeep sequence-function mapping to accurately determine the contribution of parts and phenomena involved in gene regulation.

Shape Fluctuations of Random Polyampholyte and Intrinsically Disordered Protein Sequences

Charged polymers with quenched charge sequences adopt pearl-necklace structures due to the interplay between charge disorder and thermal fluctuations. Partially globular pearl necklace conformations from random (+/−) sequences are intrinsically heterogeneous and exhibit various structures characterized by different numbers of pearls. On the basis of the molecular dynamics simulations of random model polyampholytes (PAs), we study dynamics of pearls and associated conformational heterogeneity in pearl structures. Fast nucleation/dissolution of small pearls controls the dynamics of the number of pearls, which is well described by first order kinetic equations. Most of the individual sequences we considered have a rather stable number of large pearls. Only a few sequences allow for fast switchings in the number of large pearls following complex processes (trajectories in parameter space) going through states with different numbers of pearls. Processes are most complex in the tail of the switching time distribution. The specific sequences of two intrinsically disordered proteins (IDPs) are studied along the same lines. Our study shows the resemblance in shape fluctuations between the IDP sequences and the fast-switching PA sequences.

Can the genes communicate with each other after birth? An international cross-sectional study.

Various factors contribute to the pathogenesis of a disease. These include genetic factors, family history, and some idiopathic causes. Genetic makeup has an important role in the progression of disease. This is due to mutations in genetic material, that is, deoxyribonucleic acid (DNA). This is a cross-sectional study that involved 5000 participants distributed across 250 countries. All the participants were randomly selected and asked to fill out the online survey. All the participants were fully informed about the study's purpose before providing their consent. The participants were distributed among 250 countries. Their age mean (standard deviation) is 46.7 (12.4). We discovered a significant difference between those who have genetic or congenital diseases and those who have a family history of the disease. Also, there is a statistically significant difference between the recurrence of the disease and the duration of the visits of close relatives who have the same disease. The study suggests that there might be some ways, through gene waves or the environment, in which a gene changes the expression of other genes of similar sequence in different individuals when the required period of contact is provided. In the future, this theory might explain the idiopathic nature of some diseases.

Unequal distribution of genetically-intact HIV-1 proviruses in cells expressing the immune checkpoint markers PD-1 and/or CTLA-4.

HIV-1 persists in resting CD4+ T-cells despite antiretroviral therapy (ART). Determining the cell surface markers that enrich for genetically-intact HIV-1 genomes is vital in developing targeted curative strategies. Previous studies have found that HIV-1 proviral DNA is enriched in CD4+ T-cells expressing the immune checkpoint markers programmed cell death protein-1 (PD-1) or cytotoxic T-lymphocyte associated protein-4 (CTLA-4). There has also been some success in blocking these markers in an effort to reverse HIV-1 latency. However, it remains unclear whether cells expressing PD-1 and/or CTLA-4 are enriched for genetically-intact, and potentially replication-competent, HIV-1 genomes. We obtained peripheral blood from 16 HIV-1-infected participants, and paired lymph node from four of these participants, during effective ART. Memory CD4+ T-cells from either site were sorted into four populations: PD-1-CTLA-4- (double negative, DN), PD-1+CTLA-4- (PD-1+), PD-1-CTLA-4+ (CTLA-4+) and PD-1+CTLA-4+ (double positive, DP). We performed an exploratory study using the full-length individual proviral sequencing (FLIPS) assay to identify genetically-intact and defective genomes from each subset, as well as HIV-1 genomes with specific intact open reading frames (ORFs). In peripheral blood, we observed that proviruses found within PD-1+ cells are more likely to have intact ORFs for genes such as tat, rev and nef compared to DN, CTLA-4+ and DP cells, all of which may contribute to HIV-1 persistence. Conversely, we observed that CTLA-4 expression is a marker for cells harbouring HIV-1 provirus that is more likely to be defective, containing low levels of these intact ORFs. In the lymph node, we found evidence that CTLA-4+ cells contain lower levels of HIV-1 provirus compared to the other cell subsets. Importantly, however, we observed significant participant variation in the enrichment of HIV-1 proviruses with intact genomes or specific intact ORFs across these memory CD4+ T-cell subsets, and therefore consideration of additional cellular markers will likely be needed to consistently identify cells harbouring latent, and potentially replication-competent, HIV-1.

From the Editor’s Desk...

From the Editor’s Desk...

Some orangutans acquire enamel defects at regular intervals, but not according to seasonal cycles

AbstractObjectivesDevelopmental defects of tooth enamel are associated with systemic physiological stress and have been linked to seasonal environmental factors such as rainfall, temperature, and fruit availability. Here, we evaluate whether linear enamel hypoplasia and accentuated perikymata occur with any cyclicity on lower canines and then whether cycles differ between Bornean and Sumatran orangutans.Materials and MethodsEpoxy casts of lower canines from Pongo abelii (n = 14) and P. pygmaeus (n = 33) were evaluated for perikymata and dental enamel defects. Individual developmental sequences (IDSs) were generated for each canine, tracking the position of each defect in the context of continuous perikymata (time). Autocorrelation, a form of time‐series statistical analysis was run for each canine to identify whether any cyclicity of defect expression was discernable.ResultsAutocorrelation revealed cycles of defect expression within canines, but no common cycle periodicities were identified between individuals of the same species or across species. P. pygmaeus averaged more linear enamel hypoplasia per year than P. abelli, but no other comparisons (number of defects, number of perikymata between defects, and autocorrelation analysis) revealed differences between the species.DiscussionAlthough no common patterns of defect expression were identified within or between P. abelli and P. pygmaeus, the potential for autocorrelation analysis is promising for primatological and paleoanthropological studies of seasonal phenomena.

Sequence stratigraphy of the Paleocene succession in the Kharga Oasis, Western Desert, Egypt: insights from microplankton biostratigraphy and benthic foraminifer paleoenvironments

The present work provides a comprehensive stratigraphic consideration for the Paleocene (Danian–early Thanetian) succession in the Gebel El-Teir section, Kharga Oasis, Western Desert. The study incorporates calcareous nannofossils, foraminifers (planktic and benthic), and microfacies analyses to deduce fluctuations in the relative sea level and construct a sequence stratigraphic framework. In terms of the microplankton biostratigraphy, five planktic foraminifer (P2, P3a, P4a, P4b, and P4c) and four calcareous nannofossil (NP4–NP6 and NP8) zones have been recognized. Moreover, three biostratigraphic hiatuses were recognized based on the integration between the calcareous nannofossil and planktic foraminifer bioevents. These hiatuses aided in subdividing the studied interval into four 3rd order depositional sequences (D-DS1, S-DS2, S/Th-DS3, and Th-DS4). The paleo-water-depth variations deduced from the foraminiferal and sedimentological proxies facilitate the establishment of a relative sea-level curve and, subsequently, the systems tracts of each depositional sequence were readily recognized. The shallowest water conditions (middle neritic setting) occurred during the deposition of the Danian interval. However, a much deeper paleoenvironment (deep outer neritic-upper bathyal settings) marks the Selandian/Thanetian (S/Th) transition interval. The proposed sequence stratigraphic model and the presence of some extended hiatuses attribute the primary cause for accommodation destruction/creation to the regional tectonic uplift/subsidence. However, once the basin had been formed, the relative sea level within the individual depositional sequence behaved in a manner consistent with the contemporaneous eustatic 3rd-order transgression and regression events.

Concordance of Lateralization Index for Brain Asymmetry Applied to Identify a Reliable Language Task

How can we determine which language task is relevant for examining functional hemispheric asymmetry? A problem in measuring brain asymmetry using functional magnetic resonance imaging lies in the uncertain reliability of the computed index regarding the “true” asymmetry degree. Strictly speaking, the results from the Wada test or direct cortical stimulation cannot be an exact “ground truth”, specifically for the degree of asymmetry. Therefore, we developed a method to evaluate task performance using reproducibility independent of the phenomenon of functional lateralization. Kendall’s coefficient of concordance (W) was used as the statistical measure. The underlying idea was that although various algorithms to compute the lateralization index show considerably different index values for the same data, a superior language task would reproduce similar individual ranking sequences across the algorithms; the high reproducibility of rankings across various index types would indicate a reliable task to investigate functional asymmetry regardless of index computation algorithms. Consequently, we found specificity for brain locations; a verb-generation task demonstrated the highest concordance across index types along with sufficiently high index values in the inferior frontal gyrus, whereas a narration–listening task demonstrated the highest concordance in the posterior temporo-parietal junction area.

Key changes in bovine milk immunoglobulin G during lactation: NeuAc sialylation is a hallmark of colostrum immunoglobulin G N-glycosylation.

We monitored longitudinal changes in bovine milk IgG in samples from four cows at 9 time points in between 0.5 and 28days following calving. We used peptide-centric LC-MS/MS on proteolytic digests of whole bovine milk, resulting in the combined identification of 212 individual bovine milk protein sequences, with IgG making up >50 percent of the protein content of every 0.5 d colostrum sample, which reduced to ≤3 percent in mature milk. In parallel, we analyzed IgG captured from the bovine milk samples to characterize its N-glycosylation, using dedicated methods for bottom-up glycoproteomics employing product ion-triggered hybrid fragmentation; data are available via ProteomeXchange with identifier PXD037755. The bovine milk IgG N-glycosylation profile was revealed to be very heterogeneous, consisting of >40 glycoforms. Furthermore, these N-glycosylation profiles changed substantially over the period of lactation, but consistently across the four individual cows. We identified NeuAc sialylation as the key abundant characteristic of bovine colostrum IgG, significantly decreasing in the first days of lactation, and barely detectable in mature bovine milk IgG. We also report, for the first time to our knowledge, the identification of subtype IgG3 in bovine milk, alongside the better-documented IgG1 and IgG2. The detailed molecular characteristics we describe of the bovine milk IgG, and their dynamic changes during lactation, are important not only for the fundamental understanding of the calf's immune development, but also for understanding bovine milk and its bioactive components in the context of human nutrition.