Hundreds of thousands of Rohingya refugees arrived in Bangladesh within weeks in fall 2017, quickly forming large settlements without any basic support. Humanitarian first responders provided basic necessities including food, shelter, water, sanitation, and health care. However, the challenge before them—a vast camp ravaged by diphtheria and measles superimposed on a myriad of common pathologies—was disproportionate to the resources. The needs were endless, resources finite, inadequacies abundant, and premature death inevitable. While such confines force unimaginable choices in resource allocation, they do not define the humanitarian purpose—to alleviate suffering and not allow such moral violations to become devoid of their horrifying meaning. As humanitarian workers, we maintain humanity when we care, commit, and respond to moral injustices. This refusal to abandon others in desperate situations is an attempt to rectify injustices through witnessing and solidarity. When people are left behind, we must not leave them alone.

In 2016, Rose Lamont and Tana Fishman were the first patient-clinician dyad from outside North America to attend the North American Primary Care Research Group (NAPCRG) Patient and Clinician Engagement Program workshop. They returned to New Zealand inspired and formed the Pacific People’s Health Advisory Group and a Pacific practice-based research network (PBRN). They are guided by the principles of co-design, and the Samoan research framework fa’afaletui, which emphasizes a collective approach and importance of reciprocity and relationships. Their collective inquiry aims to reduce health inequalities experienced by Pacific people in South Auckland. Their community group members and PBRN are generating research questions being answered by university-based graduate students. When they embarked, they knew not the direction in which they headed. With guidance, their community members and clinicians have led the way. By giving everyone a say in where they are going and how they get there, they are modeling what they wish to achieve—an egalitarian approach which decreases disparities for Pacific people.

PURPOSE

Operational failures are system-level errors in the supply of information, equipment, and materials to health care personnel. We aimed to review and synthesize the research literature to determine how operational failures in primary care affect the work of primary care physicians.

The American Board of Family Medicine routinely surveys its Diplomates in each national graduating cohort 3 years out of training. These data were used to characterize early career family physicians whose services include management of pregnancy and prescribing buprenorphine. A total of 261 (5.1%) respondents both provide maternity care and prescribe buprenorphine. Family physicians who care for pregnant women and also prescribe buprenorphine represented 50.4% of all buprenorphine prescribers. The family physicians in this group were trained in a small number of residency programs, with only 15 programs producing at least 25% of graduates who do this work.

PURPOSE

Anticholinergic burden (ACB), the cumulative effect of anticholinergic medications, is associated with adverse outcomes in older people but is less studied in middle-aged populations. Numerous scales exist to quantify ACB. The aims of this study were to quantify ACB in a large cohort using the 10 most common anticholinergic scales, to assess the association of each scale with adverse outcomes, and to assess overlap in populations identified by each scale.

PURPOSE

We aimed to evaluate the efficacy and safety of use of the Fasting Algorithm for Singaporeans with Type 2 Diabetes (FAST) during Ramadan.

PURPOSE

Most real-world studies on anticoagulants have been based on health insurance databases or performed in secondary care. The aim of this study was to compare safety and effectiveness between patients treated with vitamin K antagonists (VKAs) and patients treated with direct oral anticoagulants (DOACs) in a general practice setting.

PURPOSE

General practitioners (GPs) are part of the US physician workforce, but little is known about who they are, what they do, and how they differ from family physicians (FPs). We describe self-identified GPs and compare them with board-certified FPs.

PURPOSE

Although cesarean delivery is the most common surgical procedure in the United States, postoperative opioid prescribing varies greatly. We hypothesized that patient characteristics, procedural characteristics, or both would be associated with high vs low opioid use after discharge. This information could help individualize prescriptions.

PURPOSE

Online programs may help to engage patients in advance care planning in outpatient settings. We sought to implement an online advance care planning program, PREPARE (Prepare for Your Care; http://www.prepareforyourcare.org), at home and evaluate the changes in advance care planning engagement among patients attending outpatient clinics.

PURPOSE

The prognosis of older patients with dizziness in primary care is unknown. Our objective was to determine the prognosis and survival of patients with different subtypes and causes of dizziness.

Metabolic disorders are highly prevalent in kidney transplant candidates and recipients and can adversely affect post-transplant graft outcomes. Management of diabetes, hyperparathyroidism, and obesity presents distinct opportunities to optimize patients both before and after transplant as well as the ability to track objective data over time to assess a patient’s ability to partner effectively with the health care team and adhere to complex treatment regimens. Optimization of these particular disorders can most dramatically decrease the risk of surgical and cardiovascular complications post-transplant. Approximately 60% of nondiabetic patients experience hyperglycemia in the immediate post-transplant phase. Multiple risk factors have been identified related to development of new onset diabetes after transplant, and it is estimated that upward of 7%–30% of patients will develop new onset diabetes within the first year post-transplant. There are a number of medications studied in the kidney transplant population for diabetes management, and recent data and the risks and benefits of each regimen should be optimized. Secondary hyperparathyroidism occurs in most patients with CKD and can persist after kidney transplant in up to 66% of patients, despite an initial decrease in parathyroid hormone levels. Parathyroidectomy and medical management are the options for treatment of secondary hyperparathyroidism, but there is no randomized, controlled trial providing clear recommendations for optimal management, and patient-specific factors should be considered. Obesity is the most common metabolic disorder affecting the transplant population in both the pre- and post-transplant phases of care. Not only does obesity have associations and interactions with comorbid illnesses, such as diabetes, dyslipidemia, and cardiovascular disease, all of which increase morbidity and mortality post-transplant, but it also is intimately inter-related with access to transplantation for patients with kidney failure. We review these metabolic disorders and their management, including data in patients with kidney transplants.

Congenital anomalies of the kidneys and urinary tracts (CAKUT) are disorders caused by defects in the development of the kidneys and their outflow tracts. The formation of the kidneys begins at week 3 and nephrogenesis continues until week 36, therefore, the kidneys and outflow tracts are susceptible to environmental risk factors that perturb development throughout gestation. Many genes have been implicated in kidney and outflow tract development, and mutations have been identified in patients with CAKUT. In severe cases of CAKUT, when the kidneys do not form, the fetus will not survive. However, in less severe cases, the baby can survive with combined kidney and outflow tract defects or they may only be identified in adulthood. In this review, we will cover the clinical presentation of CAKUT, its epidemiology, and its long-term outcomes. We will then discuss risk factors for CAKUT, including genetic and environmental contributions. Although severe CAKUT is rare, low nephron number is a much more common disorder with its effect on kidney function increasingly apparent as a person ages. Low nephron number appears to arise by the same mechanisms as CAKUT, but it differs in terms of the magnitude of the insult and the timing of when it occurs during gestation. By understanding the causes of CAKUT and low nephron number, we can begin to identify preventive treatments and establish clinical guidelines for how these patients should be followed.

The kidney is a complex organ responsible for maintaining multiple aspects of homeostasis in the human body. The combination of distinct, yet interrelated, molecular functions across different cell types make the delineation of factors associated with loss or decline in kidney function challenging. Consequently, there has been a paucity of new diagnostic markers and treatment options becoming available to clinicians and patients in managing kidney diseases. A systems biology approach to understanding the kidney leverages recent advances in computational technology and methods to integrate diverse sets of data. It has the potential to unravel the interplay of multiple genes, proteins, and molecular mechanisms that drive key functions in kidney health and disease. The emergence of large, detailed, multilevel biologic and clinical data from national databases, cohort studies, and trials now provide the critical pieces needed for meaningful application of systems biology approaches in nephrology. The purpose of this review is to provide an overview of the current state in the evolution of the field. Recent successes of systems biology to identify targeted therapies linked to mechanistic biomarkers in the kidney are described to emphasize the relevance to clinical care and the outlook for improving outcomes for patients with kidney diseases.

Background and objectives

Bioelectrical impedance analysis (BIA) devices can help assess volume overload in patients receiving maintenance peritoneal dialysis. However, the effects of BIA on the short-term hard end points of peritoneal dialysis lack consistency. This study aimed to test whether BIA-guided fluid management could improve short-term outcomes in patients on peritoneal dialysis.

Background and objectives

Shared decision making in patients with glomerular disease remains challenging because outcomes important to patients remain largely unknown. We aimed to identify and prioritize outcomes important to patients and caregivers and to describe reasons for their choices.

Background and objectives

Tolvaptan is approved to slow kidney function decline in adults with autosomal dominant polycystic kidney disease (ADPKD) at risk of rapid progression. Because in vitro studies indicated that the tolvaptan oxobutyric acid metabolite inhibits organic anion–transporting polypeptide (OATP)1B1 and OATP1B3, United States prescribing information advises avoiding concurrent use with OATP1B1/1B3 substrates, including hepatic hydroxymethyl glutaryl–CoA reductase inhibitors (statins). This post hoc analysis of the pivotal phase 3 tolvaptan trials (Tolvaptan Efficacy and Safety in Management of Autosomal Dominant Polycystic Kidney Disease and Its Outcomes [TEMPO] 3:4 trial [NCT00428948] and Replicating Evidence of Preserved Renal Function: an Investigation of Tolvaptan Safety and Efficacy in ADPKD [REPRISE] trial [NCT02160145]) examined the safety of concurrent tolvaptan/statin use.

Background and objectives

Oxidative stress is a hallmark and mediator of CKD. Diminished antioxidant defenses are thought to be partly responsible. However, there is currently no way to prospectively assess antioxidant defenses in humans. Tin protoporphyrin (SnPP) induces mild, transient oxidant stress in mice, triggering increased expression of select antioxidant proteins (e.g., heme oxygenase 1 [HO-1], NAD[P]H dehydrogenase [quinone] 1 [NQO1], ferritin, p21). Hence, we tested the hypothesis that SnPP can also variably increase these proteins in humans and can thus serve as a pharmacologic "stress test" for gauging gene responsiveness and antioxidant reserves.

Background and objectives

Although renin-angiotensin-aldosterone system inhibition (RAASi) is a cornerstone in the treatment of children with CKD, it is sometimes discontinued when kidney function declines. We studied the reasons of RAASi discontinuation and associations between RAASi discontinuation and important risk markers of CKD progression and on eGFR decline in the Cardiovascular Comorbidity in Children with CKD study.

Background and objectives

Uromodulin is exclusively produced by tubular epithelial cells and released into urine and serum. Higher serum uromodulin has been associated with lower risk for kidney failure in Chinese patients with CKD and with lower risk for mortality in the elderly and in patients undergoing coronary angiography. We hypothesized that lower serum uromodulin is associated with mortality, cardiovascular events, and kidney failure in white patients with CKD.

Background and objectives

Large, randomized, controlled trials targeting higher hemoglobin level with erythropoiesis-stimulating agents for Western patients with CKD showed harm. However, the effect of anemia correction using erythropoiesis-stimulating agents may differ between CKD subpopulations. The Prevention of ESKD by Darbepoetin Alfa in CKD Patients with Non-diabetic Kidney Disease study, a multicenter, randomized, open-label, parallel-group study, aimed to examine the effect of targeting hemoglobin levels of 11–13 g/dl using darbepoetin alfa with reference to a low-hemoglobin target of 9–11 g/dl on kidney outcome in patients with advanced CKD without diabetes in Japan.

Background and objectives

This study aimed to investigate the association between in-hospital trajectories of serum sodium and risk of in-hospital and 1-year mortality in patients in hospital.

Metabolic reprogramming is critical for the polarization and function of tumor-associated macrophages (TAM) and hepatocarcinogenesis, but how this reprogramming occurs is unknown. Here, we showed that receptor-interacting protein kinase 3 (RIPK3), a central factor in necroptosis, is downregulated in hepatocellular carcinoma (HCC)–associated macrophages, which correlated with tumorigenesis and enhanced the accumulation and polarization of M2 TAMs. Mechanistically, RIPK3 deficiency in TAMs reduced reactive oxygen species and significantly inhibited caspase1-mediated cleavage of PPAR. These effects enabled PPAR activation and facilitated fatty acid metabolism, including fatty acid oxidation (FAO), and induced M2 polarization in the tumor microenvironment. RIPK3 upregulation or FAO blockade reversed the immunosuppressive activity of TAMs and dampened HCC tumorigenesis. Our findings provide molecular basis for the regulation of RIPK3-mediated, lipid metabolic reprogramming of TAMs, thus highlighting a potential strategy for targeting the immunometabolism of HCC.

Although treatment with the glucocorticoid-induced tumor necrosis factor receptor–related protein (GITR) agonistic antibody (DTA-1) has shown antitumor activity in various tumor models, the underlying mechanism is not fully understood. Here, we demonstrate that interleukin (IL)-21–producing follicular helper T (Tfh) cells play a crucial role in DTA-1–induced tumor inhibition. The administration of DTA-1 increased IL21 expression by Tfh cells in an antigen-specific manner, and this activation led to enhanced antitumor cytotoxic T lymphocyte (CTL) activity. Mice treated with an antibody that neutralizes the IL21 receptor exhibited decreased antitumor activity when treated with DTA-1. Tumor growth inhibition by DTA-1 was abrogated in Bcl6^fl/flCd4^Cre mice, which are genetically deficient in Tfh cells. IL4 was required for optimal induction of IL21-expressing Tfh cells by GITR costimulation, and c-Maf mediated this pathway. Thus, our findings identify GITR costimulation as an inducer of IL21-expressing Tfh cells and provide a mechanism for the antitumor activity of GITR agonism.

Epitopes derived from mutated cancer proteins elicit strong antitumor T-cell responses that correlate with clinical efficacy in a proportion of patients. However, it remains unclear whether the subcellular localization of mutated proteins influences the efficiency of T-cell priming. To address this question, we compared the immunogenicity of NY-ESO-1 and OVA localized either in the cytosol or in mitochondria. We showed that tumors expressing mitochondrial-localized NY-ESO-1 and OVA proteins elicit significantdly higher frequencies of antigen-specific CD8⁺ T cells in vivo. We also demonstrated that this stronger immune response is dependent on the mitochondrial location of the antigenic proteins, which contributes to their higher steady-state amount, compared with cytosolic localized proteins. Consistent with these findings, we showed that injection of mitochondria purified from B16 melanoma cells can protect mice from a challenge with B16 cells, but not with irrelevant tumors. Finally, we extended these findings to cancer patients by demonstrating the presence of T-cell responses specific for mutated mitochondrial-localized proteins. These findings highlight the utility of prioritizing epitopes derived from mitochondrial-localized mutated proteins as targets for cancer vaccination strategies.

T-cell receptor (TCR)–based therapeutic cells and agents have emerged as a new class of effective cancer therapies. These therapies work on cells that express intracellular cancer-associated proteins by targeting peptides displayed on MHC receptors. However, cross-reactivities of these agents to off-target cells and tissues have resulted in serious, sometimes fatal, adverse events. We have developed a high-throughput genetic platform (termed "PresentER") that encodes MHC-I peptide minigenes for functional immunologic assays and determines the reactivities of TCR-like therapeutic agents against large libraries of MHC-I ligands. In this article, we demonstrated that PresentER could be used to identify the on-and-off targets of T cells and TCR-mimic (TCRm) antibodies using in vitro coculture assays or binding assays. We found dozens of MHC-I ligands that were cross-reactive with two TCRm antibodies and two native TCRs and that were not easily predictable by other methods.

We assessed the contribution of IL1 signaling molecules to malignant tumor growth using IL1β^–/–, IL1α^–/–, and IL1R1^–/– mice. Tumors grew progressively in IL1R^–/– and IL1α^–/– mice but were often absent in IL1β^–/– mice. This was observed whether tumors were implanted intradermally or injected intravenously and was true across multiple distinct tumor lineages. Antibodies to IL1β prevented tumor growth in wild-type (WT) mice but not in IL1R1^–/– or IL1α^–/– mice. Antibodies to IL1α promoted tumor growth in IL1β^–/– mice and reversed the tumor-suppressive effect of anti-IL1β in WT mice. Depletion of CD8⁺ T cells and blockade of lymphocyte mobilization abrogated the IL1β^–/– tumor suppressive effect, as did crossing IL1β^–/– mice to SCID or Rag1^–/– mice. Finally, blockade of IL1β synergized with blockade of PD-1 to inhibit tumor growth in WT mice. These results suggest that IL1β promotes tumor growth, whereas IL1α inhibits tumor growth by enhancing T-cell–mediated antitumor immunity.

Merkel cell carcinoma (MCC) is often caused by persistent expression of Merkel cell polyomavirus (MCPyV) T-antigen (T-Ag). These non-self proteins comprise about 400 amino acids (AA). Clinical responses to immune checkpoint inhibitors, seen in about half of patients, may relate to T-Ag–specific T cells. Strategies to increase CD8⁺ T-cell number, breadth, or function could augment checkpoint inhibition, but vaccines to augment immunity must avoid delivery of oncogenic T-antigen domains. We probed MCC tumor-infiltrating lymphocytes (TIL) with an artificial antigen-presenting cell (aAPC) system and confirmed T-Ag recognition with synthetic peptides, HLA-peptide tetramers, and dendritic cells (DC). TILs from 9 of 12 (75%) subjects contained CD8⁺ T cells recognizing 1–8 MCPyV epitopes per person. Analysis of 16 MCPyV CD8⁺ TIL epitopes and prior TIL data indicated that 97% of patients with MCPyV⁺ MCC had HLA alleles with the genetic potential that restrict CD8⁺ T-cell responses to MCPyV T-Ag. The LT AA 70–110 region was epitope rich, whereas the oncogenic domains of T-Ag were not commonly recognized. Specific recognition of T-Ag–expressing DCs was documented. Recovery of MCPyV oncoprotein–specific CD8⁺ TILs from most tumors indicated that antigen indifference was unlikely to be a major cause of checkpoint inhibition failure. The myriad of epitopes restricted by diverse HLA alleles indicates that vaccination can be a rational component of immunotherapy if tumor immune suppression can be overcome, and the oncogenic regions of T-Ag can be modified without impacting immunogenicity.

Oncolytic virotherapy can lead to systemic antitumor immunity, but the therapeutic potential of oncolytic viruses in humans is limited due to their insufficient ability to overcome the immunosuppressive tumor microenvironment (TME). Here, we showed that locoregional oncolytic virotherapy upregulated the expression of PD-L1 in the TME, which was mediated by virus-induced type I and type II IFNs. To explore PD-1/PD-L1 signaling as a direct target in tumor tissue, we developed a novel immunotherapeutic herpes simplex virus (HSV), OVH-aMPD-1, that expressed a single-chain variable fragment (scFv) against PD-1 (aMPD-1 scFv). The virus was designed to locally deliver aMPD-1 scFv in the TME to achieve enhanced antitumor effects. This virus effectively modified the TME by releasing damage-associated molecular patterns, promoting antigen cross-presentation by dendritic cells, and enhancing the infiltration of activated T cells; these alterations resulted in antitumor T-cell activity that led to reduced tumor burdens in a liver cancer model. Compared with OVH, OVH-aMPD-1 promoted the infiltration of myeloid-derived suppressor cells (MDSC), resulting in significantly higher percentages of CD155⁺ granulocytic-MDSCs (G-MDSC) and monocytic-MDSCs (M-MDSC) in tumors. In combination with TIGIT blockade, this virus enhanced tumor-specific immune responses in mice with implanted subcutaneous tumors or invasive tumors. These findings highlighted that intratumoral immunomodulation with an OV expressing aMPD-1 scFv could be an effective stand-alone strategy to treat cancers or drive maximal efficacy of a combination therapy with other immune checkpoint inhibitors.

Vaccinia virus (VACV) is a double-stranded DNA virus that devotes a large portion of its 200 kbp genome to suppressing and manipulating the immune response of its host. Here, we investigated how targeted removal of immunomodulatory genes from the VACV genome impacted immune cells in the tumor microenvironment with the intention of improving the therapeutic efficacy of VACV in breast cancer. We performed a head-to-head comparison of six mutant oncolytic VACVs, each harboring deletions in genes that modulate different cellular pathways, such as nucleotide metabolism, apoptosis, inflammation, and chemokine and interferon signaling. We found that even minor changes to the VACV genome can impact the immune cell compartment in the tumor microenvironment. Viral genome modifications had the capacity to alter lymphocytic and myeloid cell compositions in tumors and spleens, PD-1 expression, and the percentages of virus-targeted and tumor-targeted CD8⁺ T cells. We observed that while some gene deletions improved responses in the nonimmunogenic 4T1 tumor model, very little therapeutic improvement was seen in the immunogenic HER2/neu TuBo model with the various genome modifications. We observed that the most promising candidate genes for deletion were those that interfere with interferon signaling. Collectively, this research helped focus attention on the pathways that modulate the immune response in the context of VACV oncolytic virotherapy. They also suggest that the greatest benefits to be obtained with these treatments may not always be seen in "hot tumors."

The identification of biomarkers for patient stratification is fundamental to precision medicine efforts in oncology. Here, we identified two baseline, circulating immune cell subsets associated with overall survival in patients with metastatic pancreatic cancer who were enrolled in two phase II randomized studies of GVAX pancreas and CRS-207 immunotherapy. Single-cell mass cytometry was used to simultaneously measure 38 cell surface or intracellular markers in peripheral blood mononuclear cells obtained from a phase IIa patient subcohort (N = 38). CITRUS, an algorithm for identification of stratifying subpopulations in multidimensional cytometry datasets, was used to identify single-cell signatures associated with clinical outcome. Patients with a higher abundance of CD8⁺CD45RO^–CCR7^–CD57⁺ cells and a lower abundance of CD14⁺CD33⁺CD85j⁺ cells had improved overall survival [median overall survival, range (days) 271, 43–1,247] compared with patients with a lower abundance of CD8⁺CD45RO^–CCR7^–CD57⁺ cells and higher abundance of CD14⁺CD33⁺CD85j⁺ cells (77, 24–1,247 days; P = 0.0442). The results from this prospective–retrospective biomarker analysis were validated by flow cytometry in 200 patients with pancreatic cancer enrolled in a phase IIb study (P = 0.0047). The identified immune correlates provide potential prognostic or predictive signatures that could be employed for patient stratification.

Patients with hematologic cancers have improved outcomes after treatment with bispecific antibodies that bind to CD3 on T cells and that redirect T cells toward cancer cells. However, clinical benefit against solid tumors remains to be shown. We made a bispecific antibody that targets both the common prostate tumor–specific antigen PSMA and CD3 (PMSAxCD3) and provide evidence for tumor inhibition in several preclinical solid tumor models. Mice expressing the human extracellular regions of CD3 and PSMA were generated to examine antitumor efficacy in the presence of an intact immune system and PSMA expression in normal tissues. PSMAxCD3 accumulated in PSMA-expressing tissues and tumors as detected by immuno-PET imaging. Although PSMAxCD3 induced T-cell activation and showed antitumor efficacy in mice with low tumor burden, PSMAxCD3 lost efficacy against larger solid tumors, mirroring the difficulty of treating solid tumors in the clinic. Costimulatory receptors can enhance T-cell responses. We show here that costimulation can enhance the antitumor efficacy of PSMAxCD3. In particular, 4-1BB stimulation in combination with PSMAxCD3 enhanced T-cell activation and proliferation, boosted efficacy against larger tumors, and induced T-cell memory, leading to durable antitumor responses. The combination of CD3 bispecific antibodies and anti-4-1BB costimulation represents a therapeutic approach for the treatment of solid tumors.

The requisites for protein translation in T cells are poorly understood and how translation shapes the antitumor efficacy of T cells is unknown. Here we demonstrated that IL15-conditioned T cells were primed by the metabolic energy sensor AMP-activated protein kinase to undergo diminished translation relative to effector T cells. However, we showed that IL15-conditioned T cells exhibited a remarkable capacity to enhance their protein translation in tumors, which effector T cells were unable to duplicate. Studying the modulation of translation for applications in cancer immunotherapy revealed that direct ex vivo pharmacologic inhibition of translation elongation primed robust T-cell antitumor immunity. Our work elucidates that altering protein translation in CD8⁺ T cells can shape their antitumor capability.

We conducted a 7-year case–control study of people ≥30 years of age on the prevalence of influenza, gastroenteritis, hepatitis, and pneumonia infections to indirectly examine whether these infections correlated to malignant cancer formation. Data were extracted from a large medical claims database of a Japanese social health insurance system; the case group included 2,354 people with their first cancer diagnosis in the 7th year of this study, and the control group included 48,395 people with no cancer diagnosis by the 7th year. The yearly prevalence rates of influenza, gastroenteritis, hepatitis, and pneumonia infections increased throughout the study period. Age-adjusted ORs and 95% confidence intervals (CI) in cases 1 year before cancer detection were significantly higher—for influenza 1.29 (95% CI, 1.14–1.46), for gastroenteritis 1.60 (95% CI, 1.41–1.82), for hepatitis 3.38 (95% CI, 2.12–5.37), for pneumonia 2.36 (95% CI, 1.79–3.13), and for any of these four diseases 1.55 (95% CI, 1.40–1.70). In influenza infections, significant ORs were found only in the 2nd and 6th years before cancer diagnosis. For each cancer site, an increased rate of infection prior to cancer diagnosis was observed. Here, we showed that increased infections during the precancerous stage, a possible surrogate for tumor-induced immune suppression, correlated to eventual cancer development.

Fnr is a transcriptional regulator that controls the expression of a variety of genes in response to oxygen limitation in bacteria. Genome sequencing revealed four genes (fnr1, fnr3, fnr5, and fnr7) coding for Fnr proteins in Paenibacillus polymyxa WLY78. Fnr1 and Fnr3 showed more similarity to each other than to Fnr5 and Fnr7. Also, Fnr1 and Fnr3 exhibited high similarity with Bacillus cereus Fnr and Bacillus subtilis Fnr in sequence and structures. Both the aerobically purified His-tagged Fnr1 and His-tagged Fnr3 in Escherichia coli could bind to the specific DNA promoter. Deletion analysis showed that the four fnr genes, especially fnr1 and fnr3, have significant impacts on growth and nitrogenase activity. Single deletion of fnr1 or fnr3 led to a 50% reduction in nitrogenase activity, and double deletion of fnr1 and fnr3 resulted to a 90% reduction in activity. Genome-wide transcription analysis showed that Fnr1 and Fnr3 indirectly activated expression of nif (nitrogen fixation) genes and Fe transport genes under anaerobic conditions. Fnr1 and Fnr3 inhibited expression of the genes involved in the aerobic respiratory chain and activated expression of genes responsible for anaerobic electron acceptor genes.

IMPORTANCE The members of the nitrogen-fixing Paenibacillus spp. have great potential to be used as a bacterial fertilizer in agriculture. However, the functions of the fnr gene(s) in nitrogen fixation and other metabolisms in Paenibacillus spp. are not known. Here, we found that in P. polymyxa WLY78, Fnr1 and Fnr3 were responsible for regulation of numerous genes in response to changes in oxygen levels, but Fnr5 and Fnr7 exhibited little effect. Fnr1 and Fnr3 indirectly or directly regulated many types of important metabolism, such as nitrogen fixation, Fe uptake, respiration, and electron transport. This study not only reveals the function of the fnr genes of P. polymyxa WLY78 in nitrogen fixation and other metabolisms but also will provide insight into the evolution and regulatory mechanisms of fnr in Paenibacillus.

This study investigated the effects of long-term soil fertilization on the composition and potential for phosphorus (P) and nitrogen (N) cycling of bacterial communities associated with hyphae of the P-solubilizing fungus Penicillium canescens. Using a baiting approach, hyphosphere bacterial communities were recovered from three soils that had received long-term amendment in the field with mineral or mineral plus organic fertilizers. P. canescens hyphae recruited bacterial communities with a decreased diversity and an increased abundance of Proteobacteria relative to what was observed in soil communities. As core bacterial taxa, Delftia and Pseudomonas spp. were present in all hyphosphere samples irrespective of soil fertilization. However, the type of fertilization showed significant impacts on the diversity, composition, and distinctive taxa/operational taxonomic units (OTUs) of hyphosphere communities. The soil factors P (Olsen method), exchangeable Mg, exchangeable K, and pH were important for shaping soil and hyphosphere bacterial community compositions. An increased relative abundance of organic P metabolism genes was found in hyphosphere communities from soil that had not received P fertilizers, which could indicate P limitation near the fungal hyphae. Additionally, P. canescens hyphae recruited bacterial communities with a higher abundance of N fixation genes than found in soil communities, which might imply a role of hyphosphere communities for fungal N nutrition. Furthermore, the relative abundances of denitrification genes were greater in several hyphosphere communities, indicating an at least partly anoxic microenvironment with a high carbon-to-N ratio around the hyphae. In conclusion, soil fertilization legacy shapes P. canescens hyphosphere microbiomes and their functional potential related to P and N cycling.

IMPORTANCE P-solubilizing Penicillium strains are introduced as biofertilizers to agricultural soils to improve plant P nutrition. Currently, little is known about the ecology of these biofertilizers, including their interactions with other soil microorganisms. This study shows that communities dominated by Betaproteobacteria and Gammaproteobacteria colonize P. canescens hyphae in soil and that the compositions of these communities depend on the soil conditions. The potential of these communities for N and organic P cycling is generally higher than that of soil communities. The high potential for organic P metabolism might complement the ability of the fungus to solubilize inorganic P, and it points to the hyphosphere as a hot spot for P metabolism. Furthermore, the high potential for N fixation could indicate that P. canescens recruits bacteria that are able to improve its N nutrition. Hence, this community study identifies functional groups relevant for the future optimization of next-generation biofertilizer consortia for applications in soil.