Shear force enhances adhesion of Pseudomonas aeruginosa by counteracting pilus-driven surface departure.

Fluid flow is thought to prevent bacterial adhesion, but some bacteria use adhesins with catch bond properties to enhance adhesion under high shear forces. However, many studies on bacterial adhesion either neglect the influence of shear force or use shear forces that are not typically found in natural systems. In this study, we use microfluidics and single-cell imaging to examine how the human pathogen Pseudomonas aeruginosa interacts with surfaces when exposed to shear forces typically found in the human body (0.1 pN to 10 pN). Through cell tracking, we demonstrate that the angle between the cell and the surface predicts if a cell will depart the surface. We discover that at lower shear forces, type IV pilus retraction tilts cells away from the surface, promoting surface departure. Conversely, we show that higher shear forces counterintuitively enhance adhesion by counteracting type IV pilus retraction-dependent cell tilting. Thus, our results reveal that P. aeruginosa exhibits behavior reminiscent of a catch bond, without having a specific adhesin that is enhanced by force. Instead, P. aeruginosa couples type IV pilus dynamics and cell geometry to tune adhesion to its mechanical environment, which likely provides a benefit in dynamic host environments.

Shear rate sensitizes bacterial pathogens to H2O2 stress.

Cells regularly experience fluid flow in natural systems. However, most experimental systems rely on batch cell culture and fail to consider the effect of flow-driven dynamics on cell physiology. Using microfluidics and single-cell imaging, we discover that the interplay of physical shear rate (a measure of fluid flow) and chemical stress trigger a transcriptional response in the human pathogen Pseudomonas aeruginosa. In batch cell culture, cells protect themselves by quickly scavenging the ubiquitous chemical stressor hydrogen peroxide (H2O2) from the media. In microfluidic conditions, we observe that cell scavenging generates spatial gradients of H2O2. High shear rates replenish H2O2, abolish gradients, and generate a stress response. Combining mathematical simulations and biophysical experiments, we find that flow triggers an effect like "wind-chill" that sensitizes cells to H2O2 concentrations 100 to 1,000 times lower than traditionally studied in batch cell culture. Surprisingly, the shear rate and H2O2 concentration required to generate a transcriptional response closely match their respective values in the human bloodstream. Thus, our results explain a long-standing discrepancy between H2O2 levels in experimental and host environments. Finally, we demonstrate that the shear rate and H2O2 concentration found in the human bloodstream trigger gene expression in the blood-relevant human pathogen Staphylococcus aureus, suggesting that flow sensitizes bacteria to chemical stress in natural environments.

Light Color Regulation of Photosynthetic Antennae Biogenesis in Marine Phytoplankton.

Photosynthesis in the world's oceans is primarily conducted by phytoplankton, microorganisms that use many different pigments for light capture. Synechococcus is a unicellular cyanobacterium estimated to be the second most abundant marine phototroph, with a global population of 7 x 1026 cells. This group's success is partly due to the pigment diversity in their photosynthetic light harvesting antennae, which maximize photon capture for photosynthesis. Many Synechococcus isolates adjust their antennae composition in response to shifts in the blue:green ratio of ambient light. This response was named Type 4 chromatic acclimation (CA4). Research has made significant progress in understanding CA4 across scales, from its global ecological importance to its molecular mechanisms. Two forms of CA4 exist, each correlated with the occurrence of one of two distinct but related genomic islands. Several genes in these islands are differentially transcribed by the ambient blue:green light ratio. The encoded proteins control the addition of different pigments to the antennae proteins in blue versus green light, altering their absorption characteristics to maximize photon capture. These genes are regulated by several putative transcription factors also encoded in the genomic islands. Ecologically, CA4 is the most abundant of marine Synechococcus pigment types, occurring in over 40% of the population oceanwide. It predominates at higher latitudes and at depth, suggesting that CA4 is most beneficial under sub-saturating photosynthetic light irradiances. Future CA4 research will further clarify the ecological role of CA4 and the molecular mechanisms controlling this globally important form of phenotypic plasticity.

Chromatic Acclimation in Cyanobacteria: A Diverse and Widespread Process for Optimizing Photosynthesis.

Chromatic acclimation (CA) encompasses a diverse set of molecular processes that involve the ability of cyanobacterial cells to sense ambient light colors and use this information to optimize photosynthetic light harvesting. The six known types of CA, which we propose naming CA1 through CA6, use a range of molecular mechanisms that likely evolved independently in distantly related lineages of the Cyanobacteria phylum. Together, these processes sense and respond to the majority of the photosynthetically relevant solar spectrum, suggesting that CA provides fitness advantages across a broad range of light color niches. The recent discoveries of several new CA types suggest that additional CA systems involving additional light colors and molecular mechanisms will be revealed in coming years. Here we provide a comprehensive overview of the currently known types of CA and summarize the molecular details that underpin CA regulation.

Evidence for biosurfactant-induced flow in corners and bacterial spreading in unsaturated porous media.

The spread of pathogenic bacteria in unsaturated porous media, where air and liquid coexist in pore spaces, is the major cause of soil contamination by pathogens, soft rot in plants, food spoilage, and many pulmonary diseases. However, visualization and fundamental understanding of bacterial transport in unsaturated porous media are currently lacking, limiting the ability to address the above contamination- and disease-related issues. Here, we demonstrate a previously unreported mechanism by which bacterial cells are transported in unsaturated porous media. We discover that surfactant-producing bacteria can generate flows along corners through surfactant production that changes the wettability of the solid surface. The corner flow velocity is on the order of several millimeters per hour, which is the same order of magnitude as bacterial swarming, one of the fastest known modes of bacterial surface translocation. We successfully predict the critical corner angle for bacterial corner flow to occur based on the biosurfactant-induced change in the contact angle of the bacterial solution on the solid surface. Furthermore, we demonstrate that bacteria can indeed spread by producing biosurfactants in a model soil, which consists of packed angular grains. In addition, we demonstrate that bacterial corner flow is controlled by quorum sensing, the cell-cell communication process that regulates biosurfactant production. Understanding this previously unappreciated bacterial transport mechanism will enable more accurate predictions of bacterial spreading in soil and other unsaturated porous media.

Bacteria in Fluid Flow.

Bacteria thrive in environments rich in fluid flow, such as the gastrointestinal tract, bloodstream, aquatic systems, and the urinary tract. Despite the importance of flow, how flow affects bacterial life is underappreciated. In recent years, the combination of approaches from biology, physics, and engineering has led to a deeper understanding of how bacteria interact with flow. Here, we highlight the wide range of bacterial responses to flow, including changes in surface adhesion, motility, surface colonization, quorum sensing, virulence factor production, and gene expression. To emphasize the diversity of flow responses, we focus our review on how flow affects four ecologically distinct bacterial species: Escherichia coli, Staphylococcus aureus, Caulobacter crescentus, and Pseudomonas aeruginosa. Additionally, we present experimental approaches to precisely study bacteria in flow, discuss how only some flow responses are triggered by shear force, and provide perspective on flow-sensitive bacterial signaling.

Medical Malpractice Lawsuits.

The tenets of a lawsuit are a deviation from the applicable standard of care that caused an injury. Elements must be addressed, including duty of care, deviation or breach of the duty to care, evidence the breach caused injury, and the identification of associated damages. Steps include consultation by a plaintiff with the attorney, pertinent records and imaging studies, and a review of the material by an expert. A complaint is filed and served upon each party. The defendant(s) must respond typically within 20 days. The parties then engage in discovery. The case may be referred to mediation, trial settlement, or dismissal.

Insemination "Fraud": Potentially Criminal Actions in Reproductive Medicine.

Case law and statutory provisions ensure marital rules of paternity apply when artificial insemination is associated with the pregnancy. Virtually all jurisdictions in the United States provide for gamete donors to remain anonymous. Much of this has been challenged with access to donor information via 23 and me. A breach of trust and a number of lawsuits involving physician provider(s) have resulted. We provide case law examples related to artificial insemination and the identification of the sperm donor. Proposed future legislation to protect patients and offspring from harm in relation to the process of donor sperm inseminations is provided.

Are Apologies a Way to Reduce Malpractice Risks?

Apologies are a means of responding to a medical error. Explanation of information related to the episode often fills a need for the patient and family to feel adequately informed. There are pros and cons related to the apology. The American College of Physicians, the American Medical Association, and the Joint Commission of the Accreditation of Health Care Organization Hospital strongly encourage practitioners to disclose when an error or complication occurs. Apologies can be admissible in the courtroom and much of this is state dependent. An apology will be an integral part of the clinician's armamentarium.

Functional Connectivity in Infancy and Toddlerhood Predicts Long-Term Language and Preliteracy Outcomes.

Functional connectivity (FC) techniques can delineate brain organization as early as infancy, enabling the characterization of early brain characteristics associated with subsequent behavioral outcomes. Previous studies have identified specific functional networks in infant brains that underlie cognitive abilities and pathophysiology subsequently observed in toddlers and preschoolers. However, it is unknown whether and how functional networks emerging within the first 18 months of life contribute to the development of higher order, complex functions of language/literacy at school-age. This 5-year longitudinal imaging project starting in infancy, utilized resting-state functional magnetic resonance imaging and demonstrated prospective associations between FC in infants/toddlers and subsequent language and foundational literacy skills at 6.5 years old. These longitudinal associations were shown independently of key environmental influences and further present in a subsample of infant imaging data (≤12 months), suggesting early emerged functional networks specifically linked to high-order language and preliteracy skills. Moreover, emergent language skills in infancy and toddlerhood contributed to the prospective associations, implicating a role of early linguistic experiences in shaping the FC correlates of long-term oral language skills. The current results highlight the importance of functional organization established in infancy and toddlerhood as a neural scaffold underlying the learning process of complex cognitive functions.

Interplay between differentially expressed enzymes contributes to light color acclimation in marine Synechococcus.

Marine Synechococcus, a globally important group of cyanobacteria, thrives in various light niches in part due to its varied photosynthetic light-harvesting pigments. Many Synechococcus strains use a process known as chromatic acclimation to optimize the ratio of two chromophores, green-light-absorbing phycoerythrobilin (PEB) and blue-light-absorbing phycourobilin (PUB), within their light-harvesting complexes. A full mechanistic understanding of how Synechococcus cells tune their PEB to PUB ratio during chromatic acclimation has not yet been obtained. Here, we show that interplay between two enzymes named MpeY and MpeZ controls differential PEB and PUB covalent attachment to the same cysteine residue. MpeY attaches PEB to the light-harvesting protein MpeA in green light, while MpeZ attaches PUB to MpeA in blue light. We demonstrate that the ratio of mpeY to mpeZ mRNA determines if PEB or PUB is attached. Additionally, strains encoding only MpeY or MpeZ do not acclimate. Examination of strains of Synechococcus isolated from across the globe indicates that the interplay between MpeY and MpeZ uncovered here is a critical feature of chromatic acclimation for marine Synechococcus worldwide.

The relationship between biological and psychosocial risk factors and resting-state functional connectivity in 2-month-old Bangladeshi infants: A feasibility and pilot study.

Childhood poverty has been associated with structural and functional alterations in the developing brain. However, poverty does not alter brain development directly, but acts through associated biological or psychosocial risk factors (e.g. malnutrition, family conflict). Yet few studies have investigated risk factors in the context of infant neurodevelopment, and none have done so in low-resource settings such as Bangladesh, where children are exposed to multiple, severe biological and psychosocial hazards. In this feasibility and pilot study, usable resting-state fMRI data were acquired in infants from extremely poor (n = 16) and (relatively) more affluent (n = 16) families in Dhaka, Bangladesh. Whole-brain intrinsic functional connectivity (iFC) was estimated using bilateral seeds in the amygdala, where iFC has shown susceptibility to early life stress, and in sensory areas, which have exhibited less susceptibility to early life hazards. Biological and psychosocial risk factors were examined for associations with iFC. Three resting-state networks were identified in within-group brain maps: medial temporal/striatal, visual, and auditory networks. Infants from extremely poor families compared with those from more affluent families exhibited greater (i.e. less negative) iFC in precuneus for amygdala seeds; however, no group differences in iFC were observed for sensory area seeds. Height-for-age, a proxy for malnutrition/infection, was not associated with amygdala/precuneus iFC, whereas prenatal family conflict was positively correlated. Findings suggest that it is feasible to conduct infant fMRI studies in low-resource settings. Challenges and practical steps for successful implementations are discussed.

Self-regulating genomic island encoding tandem regulators confers chromatic acclimation to marine Synechococcus.

The evolutionary success of marine Synechococcus, the second-most abundant phototrophic group in the marine environment, is partly attributable to this group's ability to use the entire visible spectrum of light for photosynthesis. This group possesses a remarkable diversity of light-harvesting pigments, and most of the group's members are orange and pink because of their use of phycourobilin and phycoerythrobilin chromophores, which are attached to antennae proteins called phycoerythrins. Many strains can alter phycoerythrin chromophore ratios to optimize photon capture in changing blue-green environments using type IV chromatic acclimation (CA4). Although CA4 is common in most marine Synechococcus lineages, the regulation of this process remains unexplored. Here, we show that a widely distributed genomic island encoding tandem master regulators named FciA (for type four chromatic acclimation island) and FciB plays a central role in controlling CA4. FciA and FciB have diametric effects on CA4. Interruption of fciA causes a constitutive green light phenotype, and interruption of fciB causes a constitutive blue light phenotype. These proteins regulate all of the molecular responses occurring during CA4, and the proteins' activity is apparently regulated posttranscriptionally, although their cellular ratio appears to be critical for establishing the set point for the blue-green switch in ecologically relevant light environments. Surprisingly, FciA and FciB coregulate only three genes within the Synechococcus genome, all located within the same genomic island as fciA and fciB These findings, along with the widespread distribution of strains possessing this island, suggest that horizontal transfer of a small, self-regulating DNA region has conferred CA4 capability to marine Synechococcus throughout many oceanic areas.

X-linked TEX11 mutations, meiotic arrest, and azoospermia in infertile men.

BACKGROUND: The genetic basis of nonobstructive azoospermia is unknown in the majority of infertile men. METHODS: We performed array comparative genomic hybridization testing in blood samples obtained from 15 patients with azoospermia, and we performed mutation screening by means of direct Sanger sequencing of the testis-expressed 11 gene (TEX11) open reading frame in blood and semen samples obtained from 289 patients with azoospermia and 384 controls. RESULTS: We identified a 99-kb hemizygous loss on chromosome Xq13.2 that involved three TEX11 exons. This loss, which was identical in 2 patients with azoospermia, predicts a deletion of 79 amino acids within the meiosis-specific sporulation domain SPO22. Our subsequent mutation screening showed five novel TEX11 mutations: three splicing mutations and two missense mutations. These mutations, which occurred in 7 of 289 men with azoospermia (2.4%), were absent in 384 controls with normal sperm concentrations (P=0.003). Notably, five of those TEX11 mutations were detected in 33 patients (15%) with azoospermia who received a diagnosis of azoospermia with meiotic arrest. Meiotic arrest in these patients resembled the phenotype of Tex11-deficient male mice. Immunohistochemical analysis showed specific cytoplasmic TEX11 expression in late spermatocytes, as well as in round and elongated spermatids, in normal human testes. In contrast, testes of patients who had azoospermia with TEX11 mutations had meiotic arrest and lacked TEX11 expression. CONCLUSIONS: In our study, hemizygous TEX11 mutations were a common cause of meiotic arrest and azoospermia in infertile men. (Funded by the National Institutes of Health and others.).

Chromosomal instability in women with primary ovarian insufficiency.

STUDY QUESTION: What is the prevalence of somatic chromosomal instability among women with idiopathic primary ovarian insufficiency (POI)? SUMMARY ANSWER: A subset of women with idiopathic POI may have functional impairment in DNA repair leading to chromosomal instability in their soma. WHAT IS KNOWN ALREADY: The formation and repair of DNA double-strand breaks during meiotic recombination are fundamental processes of gametogenesis. Oocytes with compromised DNA integrity are susceptible to apoptosis which could trigger premature ovarian aging and accelerated wastage of the human follicle reserve. Genomewide association studies, as well as whole exome sequencing, have implicated multiple genes involved in DNA damage repair. However, the prevalence of defective DNA damage repair in the soma of women with POI is unknown. STUDY DESIGN, SIZE, DURATION: In total, 46 women with POI and 15 family members were evaluated for excessive mitomycin-C (MMC)-induced chromosome breakage. Healthy fertile females (n = 20) and two lymphoblastoid cell lines served as negative and as positive controls, respectively. PARTICIPANTS/MATERIALS, SETTING, METHODS: We performed a pilot functional study utilizing MMC to assess chromosomal instability in the peripheral blood of participants. A high-resolution array comparative genomic hybridization (aCGH) was performed on 16 POI patients to identify copy number variations (CNVs) for a set of 341 targeted genes implicated in DNA repair. MAIN RESULTS AND THE ROLE OF CHANCE: Array CGH revealed three POI patients (3/16, 18.8%) with pathogenic CNVs. Excessive chromosomal breakage suggestive of a constitutional deficiency in DNA repair was detected in one POI patient with the 16p12.3 duplication. In two patients with negative chromosome breakage analysis, aCGH detected a Xq28 deletion comprising the Centrin EF-hand Protein 2 (CETN2) and HAUS Augmin Like Complex Subunit 7 (HAUS7) genes essential for meiotic DNA repair, and a duplication in the 3p22.2 region comprising a part of the ATPase domain of the MutL Homolog 1 (MLH1) gene. LIMITATIONS REASONS FOR CAUTION: Peripheral lymphocytes, used as a surrogate tissue to quantify induced chromosome damage, may not be representative of all the affected tissues. Another limitation pertains to the MMC assay which detects homologous repair pathway defects and does not test deficiencies in other DNA repair pathways. WIDER IMPLICATIONS OF THE FINDINGS: Our results provide evidence for functional impairment of DNA repair in idiopathic POI, which may predispose the patients to other DNA repair-related conditions such as accelerated aging and/or cancer susceptibility. STUDY FUNDING/COMPETING INTEREST(S): Funding was provided by the National Institute of Child Health and Human Development. There were no competing interests to declare.

High quality RNA in semen and sperm: isolation, analysis and potential application in clinical testing.

PURPOSE: Male infertility is a complex health condition. To our knowledge there are no molecular biomarkers of male infertility. Sperm RNA is a potential biomarker for detecting sperm abnormalities and viability at infertility clinics. However, RNA use is hindered by its inconsistent quantity, quality, multiple cell types in semen and condensed sperm structure. MATERIALS AND METHODS: We tested the usefulness of high quality RNA isolated from mature sperm and whole semen by our protocol, which reduces RNA degradation by maintaining semen and protocol components at 37 C and decreasing processing time. We isolated RNA from 83 whole semen samples, 18 samples of motile sperm prepared by the swim-up protocol and 18 of sperm prepared by the standard Percoll gradient method. RESULTS: Electrophoretic and spectral analysis of RNA revealed high quality 18S and 28S rRNAs in 71 of 83 whole semen samples (86%) and 15 of 18 mature sperm swim-up samples (83%). However, high quality RNA was isolated from only 7 of 18 Percoll gradient sperm samples (39%). Interestingly quantitative reverse transcriptase-polymerase chain reaction analysis of 4 somatic and 10 germ cell markers showed that whole semen and swim-up samples had similar RNA profiles. RNA sequencing revealed that most encoded proteins were involved in mature sperm function, regulation of DNA replication, transcription, translation, cell cycle and embryo development. CONCLUSIONS: We believe that semen and sperm specific RNAs are highly informative biomarkers for germ cell stages and somatic cell contribution. Therefore, these RNAs could be valuable diagnostic indicators of sperm survival, fertilization and early embryogenesis, and could serve as a predictor of the in vitro fertilization prognosis.

Combining multiple stressors unexpectedly blocks bacterial migration and growth.

In nature, organisms experience combinations of stressors. However, laboratory studies typically simplify reality and focus on the effects of an individual stressor. Here, we use a microfluidic approach to simultaneously provide a physical stressor (shear flow) and a chemical stressor (H 2 O 2 ) to the human pathogen Pseudomonas aeruginosa . By treating cells with levels of flow and H 2 O 2 that commonly co-occur in nature, we discover that previous reports significantly overestimate the H 2 O 2 levels required to block bacterial growth. Specifically, we establish that flow increases H 2 O 2 effectiveness 50-fold, explaining why previous studies lacking flow required much higher concentrations. Using natural H 2 O 2 levels, we identify the core H 2 O 2 regulon, characterize OxyR-mediated dynamic regulation, and dissect the redundant roles of multiple H 2 O 2 scavenging systems. By examining single-cell behavior, we serendipitously discover that the combined effects of H 2 O 2 and flow block pilus-driven surface migration. Thus, our results counter previous studies and reveal that natural levels of H 2 O 2 and flow synergize to restrict bacterial colonization and survival. By studying two stressors at once, our research highlights the limitations of oversimplifying nature and demonstrates that physical and chemical stress can combine to yield unpredictable effects.

Fluid flow overcomes antimicrobial resistance by boosting delivery.

Antimicrobial resistance is an emerging global threat to humanity. As resistance outpaces development, new perspectives are required. For decades, scientists have prioritized chemical optimization, while largely ignoring the physical process of delivery. Here, we used biophysical simulations and microfluidic experiments to explore how fluid flow delivers antimicrobials into communities of the highly resistant pathogen Pseudomonas aeruginosa . We discover that increasing flow overcomes bacterial resistance towards three chemically distinct antimicrobials: hydrogen peroxide, gentamicin, and carbenicillin. Without flow, resistant P. aeruginosa cells generate local zones of depletion by neutralizing all three antimicrobials through degradation or chemical modification. As flow increases, delivery overwhelms neutralization, allowing antimicrobials to regain effectiveness against resistant bacteria. Additionally, we discover that cells on the edge of a community shield internal cells, and cell-cell shielding is abolished in higher flow regimes. Collectively, our quantitative experiments reveal the unexpected result that physical flow and chemical dosage are equally important to antimicrobial effectiveness. Thus, our results should inspire the incorporation of flow into the discovery, development, and implementation of antimicrobials, and could represent a new strategy to combat antimicrobial resistance.

An Assessment of Business of Medicine Knowledge in Obstetrics and Gynecology Fellows: A Pilot Study.

STUDY OBJECTIVE: To identify knowledge gaps in business education among obstetrics and gynecology fellows METHODS: An online anonymous survey was distributed to obstetrics and gynecology subspecialty fellows, including pediatric and adolescent gynecology, minimally invasive gynecologic surgery, and reproductive endocrinology and infertility fellows. RESULTS: Of the 483 fellows who received the questionnaire, 159 completed the surveys, resulting in a response rate of 32.9%. A total of 80 reproductive endocrinology and infertility fellows (50.3%), 47 minimally invasive gynecologic surgery fellows (29.6%), and 32 pediatric and adolescent gynecology (20.1%) fellows completed the survey. Over half reported debt from either undergraduate or medical school (52.2%). Over half (58.5%) reported 0 hours of finance education in their residency or fellowship training. In general, fellows reported relatively higher levels of confidence in nonmedical aspects of business, such as purchasing a home (63.9%), life and disability insurance (57.2%), and making financial plans for the future (57.9%). Conversely, a large portion of fellows reported feeling "not at all confident" in business topics related to the field of medicine, including contract negotiation (24.7%), non-competes (27.1%), relative value units system-based pay (32.0%), general office practice management (58.2%), legal aspects of business (71.8%), accounting and billing (54.4%), and marketing (55.7%). CONCLUSION: Our survey demonstrates an unmet demand among obstetrics and gynecology fellows to learn topics related to the business of medicine. Knowledge of these topics is critical for those pursuing private practice or academic medicine. Future initiatives should evaluate other subspecialties and prioritize creating a standardized education tool to better prepare trainees entering medical practice.