Glycoproteome remodeling and organelle-specific N-glycosylation accompany neutrophil granulopoiesis.

Neutrophils store microbicidal glycoproteins in cytosolic granules to fight intruding pathogens, but their granule distribution and formation mechanism(s) during granulopoiesis remain unmapped. Herein, we comprehensively profile the neutrophil N-glycoproteome with spatiotemporal resolution by analyzing four key types of intracellular organelles isolated from blood-derived neutrophils and during their maturation from bone marrow-derived progenitors using a glycomics-guided glycoproteomics approach. Interestingly, the organelles of resting neutrophils exhibited distinctive glycophenotypes including, most strikingly, highly truncated N-glycans low in α2,6-sialylation and Lewis fucosylation decorating a diverse set of microbicidal proteins (e.g., myeloperoxidase, azurocidin, neutrophil elastase) in the azurophilic granules. Excitingly, proteomics and transcriptomics data from discrete myeloid progenitor stages revealed that profound glycoproteome remodeling underpins the promyelocytic-to-metamyelocyte transition and that the glycophenotypic differences are driven primarily by dynamic changes in protein expression and less by changes within the glycosylation machinery. Notable exceptions were the oligosaccharyltransferase subunits responsible for initiation of N-glycoprotein biosynthesis that were strongly expressed in early myeloid progenitors correlating with relatively high levels of glycosylation of the microbicidal proteins in the azurophilic granules. Our study provides spatiotemporal insights into the complex neutrophil N-glycoproteome featuring intriguing organelle-specific N-glycosylation patterns formed by dynamic glycoproteome remodeling during the early maturation stages of the myeloid progenitors.

Proteomics-based mass spectrometry profiling of SARS-CoV-2 infection from human nasopharyngeal samples.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the on-going global pandemic of coronavirus disease 2019 (COVID-19) that continues to pose a significant threat to public health worldwide. SARS-CoV-2 encodes four structural proteins namely membrane, nucleocapsid, spike, and envelope proteins that play essential roles in viral entry, fusion, and attachment to the host cell. Extensively glycosylated spike protein efficiently binds to the host angiotensin-converting enzyme 2 initiating viral entry and pathogenesis. Reverse transcriptase polymerase chain reaction on nasopharyngeal swab is the preferred method of sample collection and viral detection because it is a rapid, specific, and high-throughput technique. Alternate strategies such as proteomics and glycoproteomics-based mass spectrometry enable a more detailed and holistic view of the viral proteins and host-pathogen interactions and help in detection of potential disease markers. In this review, we highlight the use of mass spectrometry methods to profile the SARS-CoV-2 proteome from clinical nasopharyngeal swab samples. We also highlight the necessity for a comprehensive glycoproteomics mapping of SARS-CoV-2 from biological complex matrices to identify potential COVID-19 markers.

Assessing water, sanitation and hygiene (WASH) practices and their association with diarrhoea in under-five children in urban Chandernagore: Community-based evidence from a small municipal corporation in Hooghly District of West Bengal, India.

The study aims to understand the relationship of childhood diarrhoea (under-five children) with water, sanitation and hygiene factors in the light of other contextual factors in an urban setting in the district of Hooghly in West Bengal, India. This primary study was carried out by SIGMA Foundation, Kolkata from 4 to 24 January 2023 across 404 households having at least one under-five child. The findings suggested that the water score was 'good' in 85.1% of the households whereas the hand hygiene score was 'good' in 14.6% of households. More than 90% of the households had piped water supply. Less than half of them treated water before consumption among which 45.3% used cloths for straining water; 59.2% of the caregivers followed safe disposal of child's faeces; 66.8% of households had no handwashing arrangement, and 30.5% had taps and wash basins for handwashing; 20.3% of the under-five children had suffered from diarrhoea in the last month before the survey and its prevalence was higher in children aged 12-23 months. Multivariate results suggested diarrhoea prevalence was lower in households that were pucca and had good water and hand hygiene scores, lower in children that had received iron fortification and whose caregivers followed safe child's stool disposal.

Hyper-truncated Asn355- and Asn391-glycans modulate the activity of neutrophil granule myeloperoxidase.

Myeloperoxidase (MPO) plays essential roles in neutrophil-mediated immunity via the generation of reactive oxidation products. Complex carbohydrates decorate MPO at discrete sites, but their functional relevance remains elusive. To this end, we have characterised the structure-biosynthesis-activity relationship of neutrophil MPO (nMPO). Mass spectrometry demonstrated that nMPO carries both characteristic under-processed and hyper-truncated glycans. Occlusion of the Asn355/Asn391-glycosylation sites and the Asn323-/Asn483-glycans, located in the MPO dimerisation zone, was found to affect the local glycan processing, thereby providing a molecular basis of the site-specific nMPO glycosylation. Native mass spectrometry, mass photometry and glycopeptide profiling revealed significant molecular complexity of diprotomeric nMPO arising from heterogeneous glycosylation, oxidation, chlorination and polypeptide truncation variants and a previously unreported low-abundance monoprotomer. Longitudinal profiling of maturing, mature, granule-separated and pathogen-stimulated neutrophils demonstrated that nMPO is dynamically expressed during granulopoiesis, unevenly distributed across granules and degranulated upon activation. We also show that proMPO-to-MPO maturation occurs during early/mid-stage granulopoiesis. While similar global MPO glycosylation was observed across conditions, the conserved Asn355-/Asn391-sites displayed elevated glycan hyper-truncation, which correlated with higher enzyme activities of MPO in distinct granule populations. Enzymatic trimming of the Asn355-/Asn391-glycans recapitulated the activity gain and showed that nMPO carrying hyper-truncated glycans at these positions exhibits increased thermal stability, polypeptide accessibility and ceruloplasmin-mediated inhibition potential relative to native nMPO. Finally, molecular modelling revealed that hyper-truncated Asn355-glycans positioned in the MPO-ceruloplasmin interface are critical for uninterrupted inhibition. Here, through an innovative and comprehensive approach, we report novel functional roles of MPO glycans, providing new insight into neutrophil-mediated immunity.

N-acetyl-ß-D-hexosaminidases mediate the generation of paucimannosidic proteins via a putative noncanonical truncation pathway in human neutrophils.

We recently discovered that human neutrophils express immunomodulatory glycoproteins carrying unusual and highly truncated paucimannosidic N-glycans (Man1-3GlcNAc2Fuc0-1), but their biosynthesis remains elusive. Guided by the well-characterized truncation pathway in invertebrates and plants in which the N-acetyl-ß-D-hexosaminidase (Hex) isoenzymes catalyze paucimannosidic protein (PMP) formation, we here set out to test if the homologous human Hex α and ß subunits encoded by HEXA and HEXB drive a similar truncation pathway in human neutrophils. To this end, we performed quantitative glycomics and glycoproteomics of several CRISPR-Cas9-edited Hex-disrupted neutrophil-like HL-60 mutants (HEXA-KO and HEXB-KO) and matching unedited cell lines. Hex disruption was validated using next-generation sequencing, enzyme-linked immunosorbent assay (ELISA), quantitative proteomics and Hex activity assays. Excitingly, all Hex-disrupted mutants displayed significantly reduced levels of paucimannosylation, particularly Man2-3GlcNAc2Fuc1, relative to unedited HL-60 suggesting that both HEXA and HEXB contribute to PMP formation via a hitherto unexplored truncation pathway in neutrophils. Quantitative N-glycomics indeed demonstrated reduced utilization of a putative noncanonical truncation pathway in favor of the canonical elongation pathway in all Hex-disrupted mutants relative to unedited controls. Quantitative glycoproteomics recapitulated the truncation-to-elongation switch in all Hex-disrupted mutants and showed a greater switch for N-glycoproteins cotrafficking with Hex to the azurophilic granules of neutrophils such as myeloperoxidase. Finally, we supported the Hex-PMP relationship by documenting that primary neutrophils isolated from an early-onset Sandhoff disease patient (HEXB-/-) displayed dramatically reduced paucimannosylation relative to neutrophils from an age-matched unaffected donor. We conclude that both human Hex α and ß mediate PMP formation via a putative noncanonical truncation pathway in neutrophils.

Understanding the gap between knowledge and practice of handwashing in rural India: evidence from a cross-sectional study.

Limited studies in India had captured the gap in knowledge and practice of handwashing in the community. This study assesses the gap in knowledge and practice of handwashing in rural India. The study was conducted across 10 districts in five states of India - Andhra Pradesh, Assam, Maharashtra, Odisha and West Bengal from December 2021 to January 2022 by the SIGMA Foundation, Kolkata in collaboration with UNICEF India. Descriptive statistics, bivariate analysis, creation of indices and multinomial logistic regression were employed. Findings demonstrated that both knowledge of different aspects of hand hygiene and practice of handwashing with soap and water (HWWS) at critical times varied by socio-economic groups and also across the districts/states. Half of the respondents used only water to wash their hands after taking meals, before serving food, whenever their hands seemed dirty and before eating or cooking. Overall, the 'HWWS knowledge index' was 0.46, whereas the 'HWWS practice index' was 0.36. The correlation coefficient between the two was 0.36. The HWWS practice index was lower than the HWWS knowledge index for 50% of the sampled households. Both HWWS knowledge and practice indices were higher among females, higher educated and younger population. The gap between handwashing practice and knowledge was also higher among females and higher educated.

Fertility transition and socioeconomic development in districts of India, 2001-2016.

The fertility-development relationship is bi-directional, context-specific, multi-phased and inconsistent over time. Indian districts provide an ideal setting to study this association due to their size, diversity and disparity in socioeconomic development. The objective of this study was to understand the association of fertility and socioeconomic development among the 640 districts of India. Data were drawn from multiple sources: Censuses of India 2001 and 2011; DLHS-2; NFHS-4; and other published sources. A district-level data file for Total Fertility Rate (TFR) and a set of developmental indices were prepared for the 640 districts for 2001 and 2016. Computation of a composite index (District Development Index, DDI), Ordinary Least Squares, Two Stage Least Squares and panel regressions were employed. By 2016, almost half of all Indian districts had attained below-replacement fertility, and 15% had a TFR of above 3.0. The DDI of India increased from 0.399 in 2001 to 0.511 by 2016 and showed large variations across districts. The correlation coefficient between TFR and DDI was -0.658 in 2001 and -0.640 in 2016. Districts with a DDI of between 0.3 and 0.6 in 2001 had experienced a fertility decline of more than 20%. The fertility-development relationship was found to be strongly negative, convex and consistent over time, but the level of association varied regionally. For any given level of DDI, fertility in 2016 was lower than in 2001; and the association was stronger in districts with a DDI below 0.45. The negative convex association between the two was prominent in the northern, central and eastern regions and the curves were flatter in the west, south and north-east. The increasing number of districts with low fertility and low development draws much attention. Some outlying districts in the north-eastern states had high TFR and high DDI (>0.6). Based on the findings, a multi-layered strategy in districts with low socioeconomic development is recommended. Additional investment in education, child health, employment generation and provisioning of contraceptives would improve the human development to achieve India's demographic goals.

Glycan analysis of human neutrophil granules implicates a maturation-dependent glycosylation machinery.

Protein glycosylation is essential to trafficking and immune functions of human neutrophils. During granulopoiesis in the bone marrow, distinct neutrophil granules are successively formed. Distinct receptors and effector proteins, many of which are glycosylated, are targeted to each type of granule according to their time of expression, a process called "targeting by timing." Therefore, these granules are time capsules reflecting different times of maturation that can be used to understand the glycosylation process during granulopoiesis. Herein, neutrophil subcellular granules were fractionated by Percoll density gradient centrifugation, and N- and O-glycans present in each compartment were analyzed by LC-MS. We found abundant paucimannosidic N-glycans and lack of O-glycans in the early-formed azurophil granules, whereas the later-formed specific and gelatinase granules and secretory vesicles contained complex N- and O-glycans with remarkably elongated N-acetyllactosamine repeats with Lewis epitopes. Immunoblotting and histochemical analysis confirmed the expression of Lewis X and sialyl-Lewis X in the intracellular granules and on the cell surface, respectively. Many glycans identified are unique to neutrophils, and their complexity increased progressively from azurophil granules to specific granules and then to gelatinase granules, suggesting temporal changes in the glycosylation machinery indicative of "glycosylation by timing" during granulopoiesis. In summary, this comprehensive neutrophil granule glycome map, the first of its kind, highlights novel granule-specific glycosylation features and is a crucial first step toward a better understanding of the mechanisms regulating protein glycosylation during neutrophil granulopoiesis and a more detailed understanding of neutrophil biology and function.

The Arabidopsis SUMO E3 ligase SIZ1 mediates the temperature dependent trade-off between plant immunity and growth.

Increased ambient temperature is inhibitory to plant immunity including auto-immunity. SNC1-dependent auto-immunity is, for example, fully suppressed at 28°C. We found that the Arabidopsis sumoylation mutant siz1 displays SNC1-dependent auto-immunity at 22°C but also at 28°C, which was EDS1 dependent at both temperatures. This siz1 auto-immune phenotype provided enhanced resistance to Pseudomonas at both temperatures. Moreover, the rosette size of siz1 recovered only weakly at 28°C, while this temperature fully rescues the growth defects of other SNC1-dependent auto-immune mutants. This thermo-insensitivity of siz1 correlated with a compromised thermosensory growth response, which was independent of the immune regulators PAD4 or SNC1. Our data reveal that this high temperature induced growth response strongly depends on COP1, while SIZ1 controls the amplitude of this growth response. This latter notion is supported by transcriptomics data, i.e. SIZ1 controls the amplitude and timing of high temperature transcriptional changes including a subset of the PIF4/BZR1 gene targets. Combined our data signify that SIZ1 suppresses an SNC1-dependent resistance response at both normal and high temperatures. At the same time, SIZ1 amplifies the dark and high temperature growth response, likely via COP1 and upstream of gene regulation by PIF4 and BRZ1.

Structural basis for the recognition of nectin-like protein-5 by the human-activating immune receptor, DNAM-1.

Nectin and nectin-like (Necl) adhesion molecules are broadly overexpressed in a wide range of cancers. By binding to these adhesion molecules, the immunoreceptors DNAX accessory molecule-1 (DNAM-1), CD96 molecule (CD96), and T-cell immunoreceptor with Ig and ITIM domains (TIGIT) play a crucial role in regulating the anticancer activities of immune effector cells. However, within this axis, it remains unclear how DNAM-1 recognizes its cognate ligands. Here, we determined the structure of human DNAM-1 in complex with nectin-like protein-5 (Necl-5) at 2.8 Å resolution. Unexpectedly, we found that the two extracellular domains (D1-D2) of DNAM-1 adopt an unconventional "collapsed" arrangement that is markedly distinct from those in other immunoglobulin-based immunoreceptors. The DNAM-1/Necl-5 interaction was underpinned by conserved lock-and-key motifs located within their respective D1 domains, but also included a distinct interface derived from DNAM-1 D2. Mutation of the signature DNAM-1 "key" motif within the D1 domain attenuated Necl-5 binding and natural killer cell-mediated cytotoxicity. Altogether, our results have implications for understanding the binding mode of an immune receptor family that is emerging as a viable candidate for cancer immunotherapy.

Pattern and correlates of out-of-pocket payment (OOP) on female sterilization in India, 1990-2014.

BACKGROUND: Large scale public investment in family welfare programme has made female sterilization a free service in public health centers in India. Besides, it also provides financial compensation to acceptors. Despite these interventions, the use of contraception from private health centers has increased over time, across states and socio-economic groups in India. Though many studies have examined trends, patterns, and determinants of female sterilization services, studies on out-of-pocket payment (OOP) and compensations on sterilisation are limited in India. This paper examines the trends and variations in out-of-pocket payment (OOP) and compensations associated with female sterilization in India. METHODS: Data from the National Family Health Survey - 4, 2015-16 was used for the analyses. A composite variable based on compensation received and amount paid by users was computed and categorized into four distinct groups. Multivariate analyses were used to understand the significant predictors of OOP of female sterilization. RESULTS: Public health centers continued to be the major providers of female sterilization services; nearly 77.8% had availed themselves of free sterilization and 61.6% had received compensation for female sterilization. About two-fifths of the women in the economically well-off state like Kerala and one-third of the women in a poor state like Bihar had paid but did not receive any compensation for female sterilization. The OOP on female sterilization varies from 70 to 79% across India. The OOP on female sterilization was significantly higher among the educated and women belonging to the higher wealth quintile linking OOP to ability to pay for better quality of care. CONCLUSION: Public sector investment in family planning is required to provide free or subsidized provision of family welfare services, especially to women from a poor household. Improving the quality of female sterilization services in public health centers and rationalizing the compensation may extend the reach of family planning services in India.

Rural-urban differentials in fertility levels and fertility preferences in West Bengal, India: a district-level analysis.

Fertility in West Bengal is one of the lowest in India, and this relies heavily on the use of traditional methods of contraception. Social scientists and demographers have pointed to the historical role of the diffusion process of adhering to a small family size. The Total Fertility Rate (TFR) in Kolkata district, the state capital, is the lowest in the country, and has been a centre of low fertility historically. However, stark differences in rural-urban fertility rates have existed over the last few decades in West Bengal, but these have now started to narrow. This study aimed to capture the macro-level rural-urban differences in fertility levels and preferences in the West Bengal, and understand how socioeconomic factors affect these. Data were drawn from the Census of India (2011) and NFHS-4 (2015-16). Using census data and the Reverse-Surviving Method, the TFR of West Bengal was estimated to be 1.9, varying between 2.1 and 1.7 in rural and urban areas. The rural-urban gap in the district-level fertility rates was prominent, specifically in districts with higher levels of fertility. Kolkata, Hugli and North Twenty-Four Parganas had the lowest-low fertility (TFR = <1.5). Fewer than half of women with only one living child wanted further children, and this was somewhat higher in rural areas. Around 40% of women had achieved their desired number of children. However, a substantial proportion (43.1%) had a lower number of children than desired, varying between 45.9% and 41.7% in urban and rural areas, respectively. Contraception use, female education and age at marriage, along with the other socioeconomic factors, had a greater influence on rural fertility rates than on urban counterparts in the districts of West Bengal. Further research should be directed at understanding the contemporary fertility decline as well as the gap between ideal and desired number of children, specifically in those districts with very low fertility rates.

Protein Paucimannosylation Is an Enriched N-Glycosylation Signature of Human Cancers.

While aberrant protein glycosylation is a recognized characteristic of human cancers, advances in glycoanalytics continue to discover new associations between glycoproteins and tumorigenesis. This glycomics-centric study investigates a possible link between protein paucimannosylation, an under-studied class of human N-glycosylation [Man1-3 GlcNAc2 Fuc0-1 ], and cancer. The paucimannosidic glycans (PMGs) of 34 cancer cell lines and 133 tissue samples spanning 11 cancer types and matching non-cancerous specimens are profiled from 467 published and unpublished PGC-LC-MS/MS N-glycome datasets collected over a decade. PMGs, particularly Man2-3 GlcNAc2 Fuc1 , are prominent features of 29 cancer cell lines, but the PMG level varies dramatically across and within the cancer types (1.0-50.2%). Analyses of paired (tumor/non-tumor) and stage-stratified tissues demonstrate that PMGs are significantly enriched in tumor tissues from several cancer types including liver cancer (p = 0.0033) and colorectal cancer (p = 0.0017) and is elevated as a result of prostate cancer and chronic lymphocytic leukaemia progression (p < 0.05). Surface expression of paucimannosidic epitopes is demonstrated on human glioblastoma cells using immunofluorescence while biosynthetic involvement of N-acetyl-ß-hexosaminidase is indicated by quantitative proteomics. This intriguing association between protein paucimannosylation and human cancers warrants further exploration to detail the biosynthesis, cellular location(s), protein carriers, and functions of paucimannosylation in tumorigenesis and metastasis.

Post-Column Make-Up Flow (PCMF) Enhances the Performance of Capillary-Flow PGC-LC-MS/MS-Based Glycomics.

Deep characterization of biologically relevant glycans remains challenging. Porous graphitized carbon-liquid chromatography tandem mass spectrometry (PGC-LC-MS/MS) enables the quantitative elucidation of glycan fine structures. However, the early PGC-LC elution of smaller glycans (tri-, tetra-, and pentasaccharides) at low organic solvent content hampers their detection. In efforts to improve the glycan profiling sensitivity and accuracy, we present a new capillary-flow PGC-LC-MS/MS-based configuration comprising a post-column make-up flow (PCMF) that supplies an ion-promoting organic solvent to separated glycans prior to their detection by MS. The analytical performance of this setup was systematically evaluated against our existing capillary-flow PGC-LC-MS/MS platform (Jensen et al., Nat. Protoc. 2012, 7, 1299). Specifically, the ion intensities and signal-to-noise ratios of various classes of nonderivatized glycans from N- and O-glycoproteins and fructooligosaccharide mixtures were compared using methanol (MeOH)-, isopropanol (IPA)-, and acetonitrile (ACN)-based PCMF at various concentrations. In particular, ACN- and IPA-based PCMF dramatically increased the signal response across all glycan types (30- to 100-fold), improved the MS/MS spectral quality, and reduced the quantitative glycoprofile variation between replicates. In particular, the detection of the early eluting glycans benefitted from the PCMF. The highest sensitivity gains were achieved with the supplements of 100% ACN and IPA (equating to 57% (v/v) net concentration at the ion source) while neither compromising the favorable PGC-LC properties including the high peak capacity and glycan isomer separation nor changing the MS detection behavior. In conclusion, PCMF-based PGC-LC-MS/MS dramatically improves the glycomics sensitivity, coverage, and quantitative accuracy not least for the difficult-to-detect early eluting and low-abundance glycans detached from N- and O-glycoproteins.

IMPACT OF SEX COMPOSITION OF LIVING CHILDREN AND COUPLES' AGREEMENT ON SUBSEQUENT FERTILITY IN INDIA.

The desire for children could be considered a reliable predictor of subsequent fertility. At the same time, the sex composition of surviving children, along with other demographic and socioeconomic factors, may affect a couple's fertility desire and, therefore, their subsequent fertility. This study examined the impact of the sex composition of living children and a couple's agreement on fertility desire on their subsequent fertility in India using data came from two rounds of nationally representative surveys: the India Human Development Survey (IHDS)-I (2004-05) and IHDS-II (2011-12). To understand which factors affect the chances of an additional pregnancy or childbirth, a random effects logistic regression model was applied to the panel data. It was found that the fertility desires of both marital partners were important in determining the chances of subsequent fertility. About 35% of the couples wanting to limit children had undergone pregnancy or childbirth, while 76% of the couples wanting more children had conceived or given birth to children. In the case of discordance between the spouses, subsequent fertility was found to remain intermediate between those agreeing to continue childbirth and those wanting to limit it. The findings also affirmed that child sex preference, specifically son preference, still persists in Indian society. More than 80% of the couples with only daughters in IHDS-I mutually wanted to have additional children, whereas in families that only had sons, the chance of a subsequent pregnancy was inversely associated with the number of sons. Strong patriarchal settings, combined with cultural and socioeconomic factors, affect the persistence of sex preference in India. Programmes aimed at increasing family planning use need to address son preference and should include components that promote the value of girl children.

TO WHAT EXTENT DO COUPLES' PRE-MARITAL COMMUNICATIONS AFFECT THEIR POST-MARITAL FERTILITY BEHAVIOUR IN INDIA?

Reproduction in India is mainly confined to within marriage. The fertility preferences of spouses will not necessarily be the same, but discussion between couples creates scope for understanding between spouses after marriage. Knowing each other's opinions facilitates decision-making on sensitive matters such as contraception use and desired family size. This study used data from the India Human Development Survey-II (2011-12), and was based on a sample of 31,276 currently married women. The aim was to understand the role of pre-marital communication, studied through the choosing of husbands, mutual communication before marriage and duration of time spouses knew each other before marriage on the fertility preferences of couples post-marriage. These preferences included contraception use, who has most say on the number of children and the gap between desired and actual number of offspring. The results showed that wives who knew their husbands or who had any kind of communication with them before marriage had a greater chance of being involved in fertility decisions. However, most fertility decisions were found to be male-driven. Wives who knew their husbands for more than a month before marriage took more decisions on number of children (27%) than those who only knew their husbands from the day of their wedding (20%). Wives were less likely to have more children/sons/daughters than desired if they had some communication with their husbands before marriage. A better understanding of fertility preferences between spouses might help to curb unwanted births through delaying or limiting births by contraception use. Families in India could encourage couples to interact before marriage so they can make collective decisions on contraception use and/or the number of children they have.

Genome-wide analysis of pectate-induced gene expression in Botrytis cinerea: identification and functional analysis of putative d-galacturonate transporters.

The fungal plant pathogen Botrytis cinerea produces a spectrum of cell wall degrading enzymes for the decomposition of host cell wall polysaccharides and the consumption of the monosaccharides that are released. Especially pectin is an abundant cell wall component, and the decomposition of pectin by B. cinerea has been extensively studied. An effective concerted action of the appropriate pectin depolymerising enzymes, monosaccharide transporters and catabolic enzymes is important for complete d-galacturonic acid utilization by B. cinerea. In this study, we performed RNA sequencing to compare genome-wide transcriptional profiles between B. cinerea cultures grown in media containing pectate or glucose as sole carbon source. Transcript levels of 32 genes that are induced by pectate were further examined in cultures grown on six different monosaccharides, by means of quantitative RT-PCR, leading to the identification of 8 genes that are exclusively induced by d-galacturonic acid. Among these, the hexose transporter encoding genes Bchxt15 and Bchxt19 were functionally characterised. The subcellular location was studied of BcHXT15-GFP and BcHXT19-GFP fusion proteins expressed under control of their native promoter, in a B. cinerea wild-type strain. Both genes are expressed during growth on d-galacturonic acid and the fusion proteins are localized in plasma membranes and intracellular vesicles. Target gene knockout analysis revealed that BcHXT15 contributes to d-galacturonic acid uptake at pH 5∼5.6. The virulence of all B. cinerea hexose transporter mutants tested was unaltered on tomato and Nicotiana benthamiana leaves.

Utilizing coordination chemistry through formation of a CuII-quinalizarin complex to manipulate cell biology: An in vitro, in silico approach.

Quinalizarin, an analogue of anthracycline anticancer agents, is an anticancer agent itself. A CuII complex was prepared and characterized by elemental analysis, UV-Vis & IR spectroscopy, mass spectrometry, EPR and DFT. The intention behind the preparation of the complex was to increase cellular uptake, compare its binding with DNA against that of quinalizarin, modulation of semiquinone formation, realization of human DNA topoisomerase I & human DNA topoisomerase II inhibition and observation of anticancer activity. While the first two attributes of complex formation lead to increased efficacy, decrease in semiquinone generation could results in a compromise with efficacy. Inhibition of human DNA topoisomerase makes up this envisaged compromise in free radical activity since the complex shows remarkable ability to disrupt activities of human DNA topoisomerase I and II. The complex unlike quinalizarin, does not catalyze flow of electrons from NADH to O2 to the extent known for quinalizarin. Hence, decrease in semiquinone or superoxide radical anion could make modified quinalizarin [as CuII complex] less efficient in free radical pathway. However, it would be less cardiotoxic and that would be advantageous to qualify it as a better anticancer agent. Although binding to calf thymus DNA was comparable to quinalizarin, it was weaker than anthracyclines. Low cost of quinalizarin could justify consideration as a substitute for anthracyclines but the study revealed IC50 of quinalizarin/CuII-quinalizarin was much higher than anthracyclines or their complexes. Even then, there is a possibility that CuII-quinalizarin could be an improved and less costly form of quinalizarin as anticancer agent.

Hydathode immunity protects the Arabidopsis leaf vasculature against colonization by bacterial pathogens.

Plants prevent disease by passively and actively protecting potential entry routes against invading microbes. For example, the plant immune system actively guards roots, wounds, and stomata. How plants prevent vascular disease upon bacterial entry via guttation fluids excreted from specialized glands at the leaf margin remains largely unknown. These so-called hydathodes release xylem sap when root pressure is too high. By studying hydathode colonization by both hydathode-adapted (Xanthomonas campestris pv. campestris) and non-adapted pathogenic bacteria (Pseudomonas syringae pv. tomato) in immunocompromised Arabidopsis mutants, we show that the immune hubs BAK1 and EDS1-PAD4-ADR1 restrict bacterial multiplication in hydathodes. Both immune hubs effectively confine bacterial pathogens to hydathodes and lower the number of successful escape events of an hydathode-adapted pathogen toward the xylem. A second layer of defense, which is dependent on the plant hormones' pipecolic acid and to a lesser extent on salicylic acid, reduces the vascular spread of the pathogen. Thus, besides glands, hydathodes represent a potent first line of defense against leaf-invading microbes.

Profound N-glycan remodelling accompanies MHC-II immunopeptide presentation.

Immunopeptidomics, the study of peptide antigens presented on the cell surface by the major histocompatibility complex (MHC), offers insights into how our immune system recognises self/non-self in health and disease. We recently discovered that hyper-processed (remodelled) N-glycans are dominant features decorating viral spike immunopeptides presented via MHC-class II (MHC-II) molecules by dendritic cells pulsed with SARS-CoV-2 spike protein, but it remains unknown if endogenous immunopeptides also undergo N-glycan remodelling. Taking a multi-omics approach, we here interrogate published MHC-II immunopeptidomics datasets of cultured monocyte-like (THP-1) and breast cancer-derived (MDA-MB-231) cell lines for overlooked N-glycosylated peptide antigens, which we compare to their source proteins in the cellular glycoproteome using proteomics and N-glycomics data from matching cell lines. Hyper-processed chitobiose core and paucimannosidic N-glycans alongside under-processed oligomannosidic N-glycans were found to prevalently modify MHC-II-bound immunopeptides isolated from both THP-1 and MDA-MB-231, while complex/hybrid-type N-glycans were (near-)absent in the immunopeptidome as supported further by new N-glycomics data generated from isolated MHC-II-bound peptides derived from MDA-MB-231 cells. Contrastingly, the cellular proteomics and N-glycomics data from both cell lines revealed conventional N-glycosylation rich in complex/hybrid-type N-glycans, which, together with the identification of key lysosomal glycosidases, suggest that MHC-II peptide antigen processing is accompanied by extensive N-glycan trimming. N-glycan remodelling appeared particularly dramatic for cell surface-located glycoproteins while less remodelling was observed for lysosomal-resident glycoproteins. Collectively, our findings indicate that both under- and hyper-processed N-glycans are prevalent features of endogenous MHC-II immunopeptides, an observation that demands further investigation to enable a better molecular-level understanding of immune surveillance.