Archival single-cell genomics reveals persistent subclones during DCIS progression.

Ductal carcinoma in situ (DCIS) is a common precursor of invasive breast cancer. Our understanding of its genomic progression to recurrent disease remains poor, partly due to challenges associated with the genomic profiling of formalin-fixed paraffin-embedded (FFPE) materials. Here, we developed Arc-well, a high-throughput single-cell DNA-sequencing method that is compatible with FFPE materials. We validated our method by profiling 40,330 single cells from cell lines, a frozen tissue, and 27 FFPE samples from breast, lung, and prostate tumors stored for 3-31 years. Analysis of 10 patients with matched DCIS and cancers that recurred 2-16 years later show that many primary DCIS had already undergone whole-genome doubling and clonal diversification and that they shared genomic lineages with persistent subclones in the recurrences. Evolutionary analysis suggests that most DCIS cases in our cohort underwent an evolutionary bottleneck, and further identified chromosome aberrations in the persistent subclones that were associated with recurrence.

Gut microbiota elicits a protective immune response against malaria transmission.

Glycosylation processes are under high natural selection pressure, presumably because these can modulate resistance to infection. Here, we asked whether inactivation of the UDP-galactose:ß-galactoside-α1-3-galactosyltransferase (α1,3GT) gene, which ablated the expression of the Galα1-3Galß1-4GlcNAc-R (α-gal) glycan and allowed for the production of anti-α-gal antibodies (Abs) in humans, confers protection against Plasmodium spp. infection, the causative agent of malaria and a major driving force in human evolution. We demonstrate that both Plasmodium spp. and the human gut pathobiont E. coli O86:B7 express α-gal and that anti-α-gal Abs are associated with protection against malaria transmission in humans as well as in α1,3GT-deficient mice, which produce protective anti-α-gal Abs when colonized by E. coli O86:B7. Anti-α-gal Abs target Plasmodium sporozoites for complement-mediated cytotoxicity in the skin, immediately after inoculation by Anopheles mosquitoes. Vaccination against α-gal confers sterile protection against malaria in mice, suggesting that a similar approach may reduce malaria transmission in humans.

A spatially resolved single-cell genomic atlas of the adult human breast.

The adult human breast is comprised of an intricate network of epithelial ducts and lobules that are embedded in connective and adipose tissue1-3. Although most previous studies have focused on the breast epithelial system4-6, many of the non-epithelial cell types remain understudied. Here we constructed the comprehensive Human Breast Cell Atlas (HBCA) at single-cell and spatial resolution. Our single-cell transcriptomics study profiled 714,331 cells from 126 women, and 117,346 nuclei from 20 women, identifying 12 major cell types and 58 biological cell states. These data reveal abundant perivascular, endothelial and immune cell populations, and highly diverse luminal epithelial cell states. Spatial mapping using four different technologies revealed an unexpectedly rich ecosystem of tissue-resident immune cells, as well as distinct molecular differences between ductal and lobular regions. Collectively, these data provide a reference of the adult normal breast tissue for studying mammary biology and diseases such as breast cancer.

A Molecular Signature in Blood Reveals a Role for p53 in Regulating Malaria-Induced Inflammation.

Immunity that controls parasitemia and inflammation during Plasmodium falciparum (Pf) malaria can be acquired with repeated infections. A limited understanding of this complex immune response impedes the development of vaccines and adjunctive therapies. We conducted a prospective systems biology study of children who differed in their ability to control parasitemia and fever following Pf infection. By integrating whole-blood transcriptomics, flow-cytometric analysis, and plasma cytokine and antibody profiles, we demonstrate that a pre-infection signature of B cell enrichment, upregulation of T helper type 1 (Th1) and Th2 cell-associated pathways, including interferon responses, and p53 activation associated with control of malarial fever and coordinated with Pf-specific immunoglobulin G (IgG) and Fc receptor activation to control parasitemia. Our hypothesis-generating approach identified host molecules that may contribute to differential clinical outcomes during Pf infection. As a proof of concept, we have shown that enhanced p53 expression in monocytes attenuated Plasmodium-induced inflammation and predicted protection from fever.

Isotype switching in human memory B cells sets intrinsic antigen-affinity thresholds that dictate antigen-driven fates.

Memory B cells (MBCs) play a critical role in protection against homologous and variant pathogen challenge by either differentiating to plasma cells (PCs) or to germinal center (GC) B cells. The human MBC compartment contains both switched IgG+ and unswitched IgM+ MBCs; however, whether these MBC subpopulations are equivalent in their response to B cell receptor cross-linking and their resulting fates is incompletely understood. Here, we show that IgG+ and IgM+ MBCs can be distinguished based on their response to κ-specific monoclonal antibodies of differing affinities. IgG+ MBCs responded only to high-affinity anti-κ and differentiated almost exclusively toward PC fates. In contrast, IgM+ MBCs were eliminated by apoptosis by high-affinity anti-κ but responded to low-affinity anti-κ by differentiating toward GC B cell fates. These results suggest that IgG+ and IgM+ MBCs may play distinct yet complementary roles in response to pathogen challenge ensuring the immediate production of high-affinity antibodies to homologous and closely related challenges and the generation of variant-specific MBCs through GC reactions.

An organismal understanding of C. elegans innate immune responses, from pathogen recognition to multigenerational resistance.

The nematode Caenorhabditis elegans has been a model for studying infection since the early 2000s and many major discoveries have been made regarding its innate immune responses. C. elegans has been found to utilize some key conserved aspects of immune responses and signaling, but new interesting features of innate immunity have also been discovered in the organism that might have broader implications in higher eukaryotes such as mammals. Some of the distinctive features of C. elegans innate immunity involve the mechanisms this bacterivore uses to detect infection and mount specific immune responses to different pathogens, despite lacking putative orthologs of many important innate immune components, including cellular immunity, the inflammasome, complement, or melanization. Even when orthologs of known immune factors exist, there appears to be an absence of canonical functions, most notably the lack of pattern recognition by its sole Toll-like receptor. Instead, recent research suggests that C. elegans senses infection by specific pathogens through contextual information, including unique products produced by the pathogen or infection-induced disruption of host physiology, similar to the proposed detection of patterns of pathogenesis in mammalian systems. Interestingly, C. elegans can also transfer information of past infection to their progeny, providing robust protection for their offspring in face of persisting pathogens, in part through the RNAi pathway as well as potential new mechanisms that remain to be elucidated. Altogether, some of these strategies employed by C. elegans share key conceptual features with vertebrate adaptive immunity, as the animal can differentiate specific microbial features, as well as propagate a form of immune memory to their offspring.

Genomic and phenotypic evolution of nematode-infecting microsporidia.

Microsporidia are a large phylum of intracellular parasites that can infect most types of animals. Species in the Nematocida genus can infect nematodes including Caenorhabditis elegans, which has become an important model to study mechanisms of microsporidia infection. To understand the genomic properties and evolution of nematode-infecting microsporidia, we sequenced the genomes of nine species of microsporidia, including two genera, Enteropsectra and Pancytospora, without any previously sequenced genomes. Core cellular processes, including metabolic pathways, are mostly conserved across genera of nematode-infecting microsporidia. Each species encodes unique proteins belonging to large gene families that are likely used to interact with host cells. Most strikingly, we observed one such family, NemLGF1, is present in both Nematocida and Pancytospora species, but not any other microsporidia. To understand how Nematocida phenotypic traits evolved, we measured the host range, tissue specificity, spore size, and polar tube length of several species in the genus. Our phylogenetic analysis shows that Nematocida is composed of two groups of species with distinct traits and that species with longer polar tubes infect multiple tissues. Together, our work details both genomic and trait evolution between related microsporidia species and provides a useful resource for further understanding microsporidia evolution and infection mechanisms.

Potentiation of plant defense by bacterial outer membrane vesicles is mediated by membrane nanodomains.

Outer membrane vesicles (OMVs) are released from the outer membranes of Gram-negative bacteria during infection and modulate host immunity during host-pathogen interactions. The mechanisms by which OMVs are perceived by plants and affect host immunity are unclear. Here, we used the pathogen Xanthomonas campestris pv. campestris to demonstrate that OMV-plant interactions at the Arabidopsis thaliana plasma membrane (PM) modulate various host processes, including endocytosis, innate immune responses, and suppression of pathogenesis by phytobacteria. The lipid phase of OMVs is highly ordered and OMVs directly insert into the Arabidopsis PM, thereby enhancing the plant PM's lipid order; this also resulted in strengthened plant defenses. Strikingly, the integration of OMVs into the plant PM is host nanodomain- and remorin-dependent. Using coarse-grained simulations of molecular dynamics, we demonstrated that OMV integration into the plant PM depends on the membrane lipid order. Our computational simulations further showed that the saturation level of the OMV lipids could fine-tune the enhancement of host lipid order. Our work unraveled the mechanisms underlying the ability of OMVs produced by a plant pathogen to insert into the host PM, alter host membrane properties, and modulate plant immune responses.

The Identification of Enteric Fever-Specific Antigens for Population-Based Serosurveillance.

BACKGROUND: Enteric fever, caused by Salmonella enterica serovars Typhi and Paratyphi A, is a major public health problem in low- and middle-income countries. Moderate sensitivity and scalability of current methods likely underestimate enteric fever burden. Determining the serological responses to organism-specific antigens may improve incidence measures. METHODS: Plasma samples were collected from blood culture-confirmed enteric fever patients, blood culture-negative febrile patients over the course of 3 months, and afebrile community controls. A panel of 17 Salmonella Typhi and Paratyphi A antigens was purified and used to determine antigen-specific antibody responses by indirect ELISAs. RESULTS: The antigen-specific longitudinal antibody responses were comparable between enteric fever patients, patients with blood culture-negative febrile controls, and afebrile community controls for most antigens. However, we found that IgG responses against STY1479 (YncE), STY1886 (CdtB), STY1498 (HlyE), and the serovar-specific O2 and O9 antigens were greatly elevated over a 3-month follow up period in S. Typhi/S. Paratyphi A patients compared to controls, suggesting seroconversion. CONCLUSIONS: We identified a set of antigens as good candidates to demonstrate enteric fever exposure. These targets can be used in combination to develop more sensitive and scalable approaches to enteric fever surveillance and generate invaluable epidemiological data for informing vaccine policies. CLINICAL TRIAL REGISTRATION: ISRCTN63006567.

Mature beyond their years: young children who escape detection of parasitemia despite living in settings of intense malaria transmission.

Despite having the highest risk of progressing to severe disease due to lack of acquired immunity, the youngest children living in areas of highly intense malaria transmission have long been observed to be infected at lower rates than older children. Whether this observation is due to reduced exposure to infectious mosquito bites from behavioral and biological factors, maternally transferred immunity, genetic factors, or enhanced innate immunity in the young child has intrigued malaria researchers for over half a century. Recent evidence suggests that maternally transferred immunity may be limited to early infancy and that the young child's own immune system may contribute to control of malarial symptoms early in life and prior to the development of more effective adaptive immunity. Prospective studies of active and passive detection of Plasmodium falciparum blood-stage infections have identified young children (<5 years old) who remain uninfected through a defined surveillance period despite living in settings of highly intense malaria transmission. Yet, little is known about the potential immunological basis for this 'aparasitemic' phenotype. In this review, we summarize the observational evidence for this phenotype in field studies and examine potential reasons why these children escape detection of parasitemia, covering factors that are either extrinsic or intrinsic to their developing immune system. We discuss the challenges of distinguishing malaria protection from lack of malaria exposure in field studies. We also identify gaps in our knowledge regarding cellular immunity in the youngest age group and propose directions that researchers may take to address these gaps.

Balanced callose and cellulose biosynthesis in Arabidopsis quorum-sensing signaling and pattern-triggered immunity.

The plant cell wall (CW) is one of the most important physical barriers that phytopathogens must conquer to invade their hosts. This barrier is a dynamic structure that responds to pathogen infection through a complex network of immune receptors, together with CW-synthesizing and CW-degrading enzymes. Callose deposition in the primary CW is a well-known physical response to pathogen infection. Notably, callose and cellulose biosynthesis share an initial substrate, UDP-glucose, which is the main load-bearing component of the CW. However, how these 2 critical biosynthetic processes are balanced during plant-pathogen interactions remains unclear. Here, using 2 different pathogen-derived molecules, bacterial flagellin (flg22) and the diffusible signal factor (DSF) produced by Xanthomonas campestris pv. campestris, we show a negative correlation between cellulose and callose biosynthesis in Arabidopsis (Arabidopsis thaliana). By quantifying the abundance of callose and cellulose under DSF or flg22 elicitation and characterizing the dynamics of the enzymes involved in the biosynthesis and degradation of these 2 polymers, we show that the balance of these 2 CW components is mediated by the activity of a ß-1,3-glucanase (BG2). Our data demonstrate balanced cellulose and callose biosynthesis during plant immune responses.

Nanoparticles as carriers of photosensitizers to improve photodynamic therapy in cancer.

Photodynamic therapy (PDT) has emerged as a promising non invasive therapeutic approach for cancer treatment, offering unique advantages over conventional treatments. The combination of light activation and photosensitizing agents allows for targeted and localized destruction of cancer cells, reducing damage to surrounding healthy tissues. In recent years, the integration of nanoparticles with PDT has garnered significant attention due to their potential to enhance therapeutic outcomes. This review article aims to provide a comprehensive overview of the current state-of-the-art in utilizing nanoparticles for photodynamic therapy in cancer treatment. We summarized various nanoparticle-based approaches, their properties, and their implications in optimizing PDT efficacy, and discussed challenges and prospects in the field.

Decoding the complexities of human malaria through systems immunology.

The complexity of the Plasmodium parasite and its life cycle poses a challenge to our understanding of the host immune response against malaria. Studying human immune responses during natural and experimental Plasmodium infections can enhance our understanding of malaria-protective immunity and inform the design of disease-modifying adjunctive therapies and next-generation malaria vaccines. Systems immunology can complement conventional approaches to facilitate our understanding of the complex immune response to the highly dynamic malaria parasite. In this review, recent studies that used systems-based approaches to evaluate human immune responses during natural and experimental Plasmodium falciparum and Plasmodium vivax infections as well as during immunization with candidate malaria vaccines are summarized and related to each other. The potential for next-generation technologies to address the current limitations of systems-based studies of human malaria are discussed.

Environmental relevance monitoring and assessment of ochreous precipitates, hydrochemistry and water sources from abandoned coal mine drainage.

This study investigated the mineralogical and chemical characteristics of ochreous precipitates and mine water samples from abandoned Upper Carboniferous hard coal mines in an extensive former mining area in western Germany. Mine water characteristics have been monitored and assessed using a multi-methodological approach. Thirteen mine water discharge locations were sampled for hydrochemical analysis, with a total of 46 water samples seasonally collected in the whole study area for stable isotopic analyses. Mineralogical composition of 13 ochreous precipitates was identified by a combination of powder X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), and field emission scanning electron microscopy (FE-SEM/EDS). Results showed that abandoned mine drainage was characterized by circumneutral pH, Eh values ranging from 163 to 269 mV, relatively low concentrations of Fe and Mn, and was dominated by HCO3- > SO42- > Cl- > NO3- and Na+ > Ca2+ > Mg2+ > K+. Goethite and ferrihydrite were the dominant precipitated Fe minerals, with traces of quartz, dolomite, and clay minerals. Some metal and metalloid elements (Mn, Al, Si, and Ti) were found in the ochreous sediments. The role of bacteria in the formation of secondary minerals was assessed with the detection of Leptothrix ochracea. The δ18O and δ2H values of mine water plotted on and close to the GMWL and LMWLs indicated local derivation from meteoric water and represented the annual mean precipitation isotopic composition. Results might help to develop strategies for the management of water resources, contaminated mine water, and public health.

Combination of Anterior and Posterior Vaginal Mesh Implants and Delorme Procedure in a 94-year-old female with Pelvic Organ Prolapse.

Pelvic organ prolapse refers to the descent of pelvic floor organs resulting from the weakening of pelvic muscles, fascia and connective tissue. The overall prevalence of pelvic organ prolapse is approximately 41%, including bladder prolapse (25%-34%), uterine prolapse (4%-14%) and rectal prolapse (13%-19%). Various methods are currently employed to repair damaged structures and improve patient symptoms, consequently enhancing their quality of life. This report focuses on a 94-year-old female diagnosed with pelvic organ prolapse, specifically Grade 3 bladder prolapse, Grade 3 uterine prolapse and complete rectal prolapse. A comprehensive surgical treatment was carried out to repair the pelvic organs on all three levels (rectum, uterus and bladder) by combining the Delorme procedure with synthetic graft implants. The surgical outcomes were good, illustrating immediate improvement in symptoms without early complications. A multispeciality approach helps functionally repair pelvic organ prolapse while preserving structural integrity.

Neither the African-Centric S47 Nor P72 Variant of TP53 Is Associated With Reduced Risk of Febrile Malaria in a Malian Cohort Study.

BACKGROUND: TP53 has been shown to play a role in inflammatory processes, including malaria. We previously found that p53 attenuates parasite-induced inflammation and predicts clinical protection to Plasmodium falciparum infection in Malian children. Here, we investigated whether p53 codon 47 and 72 polymorphisms are associated with differential risk of P. falciparum infection and uncomplicated malaria in a prospective cohort study of malaria immunity. METHODS: p53 codon 47 and 72 polymorphisms were determined by sequencing TP53 exon 4 in 631 Malian children and adults enrolled in the Kalifabougou cohort study. The effects of these polymorphisms on the prospective risk of febrile malaria, incident parasitemia, and time to fever after incident parasitemia over 6 months of intense malaria transmission were assessed using Cox proportional hazards models. RESULTS: Confounders of malaria risk, including age and hemoglobin S or C, were similar between individuals with or without p53 S47 and R72 polymorphisms. Relative to their respective common variants, neither S47 nor R72 was associated with differences in prospective risk of febrile malaria, incident parasitemia, or febrile malaria after parasitemia. CONCLUSIONS: These findings indicate that p53 codon 47 and 72 polymorphisms are not associated with protection against incident P. falciparum parasitemia or uncomplicated febrile malaria.

Accumulation of Neutrophil Phagocytic Antibody Features Tracks With Naturally Acquired Immunity Against Malaria in Children.

BACKGROUND: Studies have demonstrated the protective role of antibodies against malaria. Young children are known to be particularly vulnerable to malaria, pointing to the evolution of naturally acquired clinical immunity over time. However, whether changes in antibody functionality track with the acquisition of naturally acquired malaria immunity remains incompletely understood. METHODS: Using systems serology, we characterized sporozoite- and merozoite-specific antibody profiles of uninfected Malian children before the malaria season who differed in their ability to control parasitemia and fever following Plasmodium falciparum (Pf) infection. We then assessed the contributions of individual traits to overall clinical outcomes, focusing on the immunodominant sporozoite CSP and merozoite AMA1 and MSP1 antigens. RESULTS: Humoral immunity evolved with age, with an expansion of both magnitude and functional quality, particularly within blood-stage phagocytic antibody activity. Moreover, concerning clinical outcomes postinfection, protected children had higher antibody-dependent neutrophil activity along with higher levels of MSP1-specific IgG3 and IgA and CSP-specific IgG3 and IgG4 prior to the malaria season. CONCLUSIONS: These data point to the natural evolution of functional humoral immunity to Pf with age and highlight particular antibody Fc-effector profiles associated with the control of malaria in children, providing clues for the design of next-generation vaccines or therapeutics.

The Germin-like protein OsGER4 is involved in promoting crown root development under exogenous jasmonic acid treatment in rice.

In rice (Oryza sativa L.), crown roots (CRs) have many important roles in processes such as root system expansion, water and mineral uptake, and adaptation to environmental stresses. Phytohormones such as auxin, cytokinin, and ethylene are known to control CR initiation and development in rice. However, the role of jasmonic acid (JA) in CR development remained elusive. Here, we report that JA promotes CR development by regulating OsGER4, a rice Germin-like protein. Root phenotyping analysis revealed that exogenous JA treatment induced an increase in CR number in a concentration-dependent manner. A subsequent genome-wide association study and gene expression analyses pinpointed a strong association between the Germin-like protein OsGER4 and the increase in CR number under exogenous JA treatment. The ProGER4::GUS reporter line showed that OsGER4 is a hormone-responsive gene involved in various stress responses, mainly confined to epidermal and vascular tissues during CR primordia development and to vascular bundles of mature crown and lateral roots. Notable changes in OsGER4 expression patterns caused by the polar auxin transport inhibitor NPA support its connection to auxin signaling. Phenotyping experiments with OsGER4 knockout mutants confirmed that this gene is required for CR development under exogenous JA treatment. Overall, our results provide important insights into JA-mediated regulation of CR development in rice.

Adenovirus-α-Defensin Complexes Induce NLRP3-Associated Maturation of Human Phagocytes via Toll-Like Receptor 4 Engagement.

Intramuscular delivery of human adenovirus (HAdV)-based vaccines leads to rapid recruitment of neutrophils, which then release antimicrobial peptides/proteins (AMPs). How these AMPs influence vaccine efficacy over the subsequent 24 h is poorly understood. In this study, we asked if human neutrophil protein 1 (HNP-1), an α-defensin that influences direct and indirect innate immune responses to a range of pathogens, impacts the response of human phagocytes to three HAdV species/types (HAdV-C5, -D26, -B35). We show that HNP-1 binds to the capsids and redirects HAdV-C5, -D26, and -B35 to Toll-like receptor 4 (TLR4), which leads to internalization, an NLRP3-mediated inflammasome response, and interleukin 1 beta (IL-1ß) release. Surprisingly, IL-1ß release was not associated with notable disruption of plasma membrane integrity. These data further our understanding of HAdV vaccine immunogenicity and may provide pathways to extend the efficacy. IMPORTANCE This study examines the interactions between danger-associated molecular patterns and human adenoviruses, and their impact on vaccines. HAdVs and HNP-1 can interact, and these interactions will modify the response of antigen-presenting cells, which will influence vaccine efficacy.