OPEN leaf: an open-source cloud-based phenotyping system for tracking dynamic changes at leaf-specific resolution in Arabidopsis.

The first draft of the Arabidopsis genome was released more than 20 years ago and despite intensive molecular research, more than 30% of Arabidopsis genes remained uncharacterized or without an assigned function. This is in part due to gene redundancy within gene families or the essential nature of genes, where their deletion results in lethality (i.e., the dark genome). High-throughput plant phenotyping (HTPP) offers an automated and unbiased approach to characterize subtle or transient phenotypes resulting from gene redundancy or inducible gene silencing; however, access to commercial HTPP platforms remains limited. Here we describe the design and implementation of OPEN leaf, an open-source phenotyping system with cloud connectivity and remote bilateral communication to facilitate data collection, sharing and processing. OPEN leaf, coupled with our SMART imaging processing pipeline was able to consistently document and quantify dynamic changes at the whole rosette level and leaf-specific resolution when plants experienced changes in nutrient availability. Our data also demonstrate that VIS sensors remain underutilized and can be used in high-throughput screens to identify and characterize previously unidentified phenotypes in a leaf-specific time-dependent manner. Moreover, the modular and open-source design of OPEN leaf allows seamless integration of additional sensors based on users and experimental needs.

The effects of chromium propionate supplementation to yearling steers in a commercial feedyard on growth performance, carcass characteristics, and health.

British crossbred steers (n = 3,072; initial body weight [BW] = 358 ±â€…37 kg) were used to evaluate the effects of chromium propionate supplementation to yearling steers in a commercial feedyard on growth performance, carcass characteristics, and health. Steers were blocked by initial BW; pens were assigned randomly to one of two dietary treatments within block. Treatments, replicated in 15 pens per treatment with 75 to 135 heads per pen, included 1) control, 0 mg supplemental Cr/kg dietary dry matter (DM) (CTL); 2) 0.50 mg supplemental Cr/kg diet DM (chromium propionate; KemTRACE Chromium 0.4%, Kemin Industries, Des Moines, IA) (chromium propionate, CR). Final BW (638 vs. 641 kg), average daily gain (1.81 vs. 1.82 kg), DM intake (11.02 vs. 11.02 kg), and gain efficiency (0.164 vs. 0.165) did not differ between CTL and CR, respectively (P ≥ 0.75). No differences among treatments for hot carcass weight (407 vs. 408 kg, CTL and CR, respectively), dressing percentage, longissimus muscle area, or yield grade were observed (P ≥ 0.15). Twelfth-rib fat thickness tended (P = 0.10) to be greater for CR vs. CTL (1.55 vs. 1.29 cm, respectively). A trend (P = 0.10) for marbling score to be higher for CR vs. CTL was detected (452 vs. 440, respectively). Distribution of quality grade was similar between CR and CTL; 1.52% of carcasses graded prime (P = 0.68), and 87.2% of carcasses graded choice (P = 0.68). Respiratory morbidity was low (1.93%) and not different among treatments (P = 0.20); likewise, there was no difference in respiratory treatment rates between treatments (P ≥ 0.18). Supplementing Cr to high-performing yearling steers did not alter growth performance, carcass characteristics, or health outcomes.

Ion Exchange Biomaterials to Capture Daptomycin and Prevent Resistance Evolution in Off-Target Bacterial Populations.

Daptomycin (DAP), a cyclic anionic lipopeptide antibiotic, is among the last resorts to treat multidrug-resistant Gram-positive bacterial infections, caused by vancomycin-resistant Enterococcus faecium or methicillin-resistant Staphylococcus aureus. DAP is administered intravenously, and via biliary excretion, ∼5-10% of the intravenous DAP dose arrives in the gastrointestinal (GI) tract where it drives resistance evolution in the off-target populations of E. faecium bacteria. Previously, we have shown in vivo that the oral administration of cholestyramine, an ion exchange biomaterial (IXB) sorbent, prevents DAP treatment from enriching DAP resistance in the populations of E. faecium shed from mice. Here, we investigate the biomaterial-DAP interfacial interactions to uncover the antibiotic removal mechanisms. The IXB-mediated DAP capture from aqueous media was measured in controlled pH/electrolyte solutions and in the simulated intestinal fluid (SIF) to uncover the molecular and colloidal mechanisms of DAP removal from the GI tract. Our findings show that the IXB electrostatically adsorbs the anionic antibiotic via a time-dependent diffusion-controlled process. Unsteady-state diffusion-adsorption mass balance describes the dynamics of adsorption well, and the maximum removal capacity is beyond the electric charge stoichiometric ratio because of DAP self-assembly. This study may open new opportunities for optimizing cholestyramine adjuvant therapy to prevent DAP resistance, as well as designing novel biomaterials to remove off-target antibiotics from the GI tract.

Androgen receptor signaling in the lungs mitigates inflammation and improves the outcome of influenza in mice.

Circulating androgens can modulate immune cell activity, but the impact of androgens on viral pathogenesis remains unclear. Previous data demonstrate that testosterone reduces the severity of influenza A virus (IAV) infection in male mice by mitigating pulmonary inflammation rather than by affecting viral replication. To examine the immune responses mediated by testosterone to mitigate IAV-induced inflammation, adult male mice remained gonadally intact or were gonadectomized and treated with either placebo or androgen-filled (i.e., testosterone or dihydrotestosterone) capsules prior to sublethal IAV infection. Like intact males, treatment of gonadectomized males with androgens improved the outcome of IAV infection, which was not mediated by changes in the control of virus replication or pulmonary cytokine activity. Instead, androgens accelerated pulmonary leukocyte contraction to limit inflammation. To identify which immune cells were contracting in response to androgens, the composition of pulmonary cellular infiltrates was analyzed and revealed that androgens specifically accelerated the contraction of total pulmonary inflammatory monocytes during peak disease, as well as CD8+ T cells, IAV-specific CD8+ T numbers, cytokine production and degranulation by IAV-specific CD8+ T cells, and the influx of eosinophils into the lungs following clearance of IAV. Neither depletion of eosinophils nor adoptive transfer of CD8+ T cells could reverse the ability of testosterone to protect males against IAV suggesting these were secondary immunologic effects. The effects of testosterone on the contraction of immune cell numbers and activity were blocked by co-administration of the androgen receptor antagonist flutamide and mimicked by treatment with dihydrotestosterone, which was also able to reduce the severity of IAV in female mice. These data suggest that androgen receptor signaling creates a local pulmonary environment that promotes downregulation of detrimental inflammatory immune responses to protect against prolonged influenza disease.

Mitochondrial mRNA fragments are circularized in a human HEK cell line.

The relatively recent focus on the widespread occurrence and abundance of circular RNAs (circRNA) in the human cell nucleus has sparked an intensive interest in their existence and possible roles in cell gene expression and physiology. The presence of circRNAs in mammalian mitochondria, however, has been under-explored. Mitochondrial mRNAs differ from those produced from nuclear genes because they lack introns and are transcribed as poly-cistronic transcripts that are endonucleolytically cleaved, leaving transcripts with very small 5' and 3' UTRs. Circular RNAs have been identified in the semi-autonomous organelles of single-celled organisms and plants but their purpose has not been clearly demonstrated. The goal of our project was to test the hypothesis, processed mRNAs are circularized in vertebrate mitochondria as a necessary RNA processing step prior to translation. Mitochondrial mRNAs were isolated from the human cell line HEK293 and evidence of circularization sought by treating RNA with RNAse-R and then amplifying putative 3'-5' junction sites. Sequence results demonstrated the occurrence of mRNA circularization within each coding region of the mitochondrial genome. However, in most cases the circRNAs carried coding regions that had been truncated, suggesting they were not translatable. Quantification of the circularized versions of the mRNAs revealed they comprise a small portion (~10%) of the total mRNA. These findings demonstrate that mRNA circularization occurs in mammalian mitochondria but it does not appear to play a role in making translatable mRNAs.

Testosterone treatment of aged male mice improves some but not all aspects of age-associated increases in influenza severity.

The severity of influenza increases with age, with worse disease in aged males than females. Testosterone concentrations decline with age in males, which may impact influenza pathogenesis. Aged male mice were treated with testosterone or placebo and outcomes during influenza A virus (IAV) infection were compared with adult male mice. Aged males experienced greater morbidity and mortality than adult males, which was partially improved by testosterone treatment of aged males. Aged males cleared IAV from lungs slower than adult males, regardless of testosterone treatment. As compared with adult males, aged males experienced pulmonary, but not systemic, cytokine dysregulation, and delayed influx and contraction of IAV-specific CD8+ T cells in the lungs. Testosterone treatment in aged males partially restored pulmonary cytokine responses to levels consistent with adult males but did not alter the age-associated changes in IAV-specific CD8+ T cells. Testosterone only modestly improves outcomes of influenza in aged males.

Irradiated sporozoite vaccination induces sex-specific immune responses and protection against malaria in mice.

In both preclinical animal studies and human clinical trials, adult females tend to develop greater adaptive immune responses than males following receipt of either viral or bacterial vaccines. While there is currently no approved malaria vaccine, several anti-sporozoite vaccines, including RTS,S/AS01 and attenuated sporozoite vaccines, are in development, but the impact of sex and age on their efficacy remains undefined. To examine sex differences in the efficacy of anti-sporozoite stage malaria vaccination, adult (10 weeks of age) or juvenile (11 days of age) male and female C3H mice were twice vaccinated with irradiated transgenic Plasmodium berghei sporozoites expressing the P. falciparum circumsporozoite (CSP) protein and 45 days post boost vaccination, mice were challenged with transgenic P. berghei via mosquito bite or intradermal challenge. Immunization with irradiated sporozoites resulted in greater protection against challenge in adult females, which was associated with greater anti-CSP antibody production and avidity, as well as greater hepatic, but not splenic, CD8+ T cell IFNƴ production in adult females than adult males. No sex differences in adaptive immune responses or protection were observed in mice vaccinated prior to puberty, suggesting a role for sex steroid hormones. Depletion of testosterone in males increased, whereas rescue of testosterone decreased, anti-CSP antibody production, the number of antigen-specific CD8+ T cells isolated from the liver, and protection following parasite challenge. Conversely, depletion of sex steroids in female mice did not alter vaccine-induced responses or protection following challenge. These data suggest that elevated testosterone concentrations in males reduce adaptive immunity and contribute to sex differences in malaria vaccine efficacy.

Sex and sex steroids impact influenza pathogenesis across the life course.

Males and females differ in the outcome of influenza A virus (IAV) infections, which depends significantly on age. During a typical seasonal influenza epidemic, young children (< 10 years of age) and aged adults (65+ years of age) are at greatest risk for severe disease, and among these age groups, males tend to suffer a worse outcome from IAV infection than females. Following infection with either pandemic or outbreak strains of IAVs, females of reproductive ages (i.e., 15-49 years of age) experience a worse outcome than their male counterparts. Among females of reproductive ages, pregnancy is one factor linked to an increased risk of severe outcome of influenza, although it is not the sole factor explaining the female-preponderance of severe disease. Small animal models of influenza virus infection illustrate that inflammatory immune responses and repair of damaged tissue following IAV infection also differ between the sexes and impact the outcome of infection. There also is growing evidence that sex steroid hormones, including estrogens, progesterone, and testosterone, directly impact immune responses during IAV infection to alter outcomes. Greater consideration of the combined effects of sex and age as biological variables in epidemiological, clinical, and animal studies of influenza pathogenesis is needed.

Production of amphiregulin and recovery from influenza is greater in males than females.

BACKGROUND: Amphiregulin (AREG) is an epidermal growth factor that is a significant mediator of tissue repair at mucosal sites, including in the lungs during influenza A virus (IAV) infection. Previous research illustrates that males of reproductive ages experience less severe disease and recover faster than females following infection with IAV. METHODS: Whether males and females differentially produce and utilize AREG for pulmonary repair after IAV infection was investigated using murine models on a C57BL/6 background and primary mouse and human epithelial cell culture systems. RESULTS: Following sublethal infection with 2009 H1N1 IAV, adult female mice experienced greater morbidity and pulmonary inflammation during the acute phase of infection as well as worse pulmonary function during the recovery phase of infection than males, despite having similar virus clearance kinetics. As compared with females, AREG expression was greater in the lungs of male mice as well as in primary respiratory epithelial cells derived from mouse and human male donors, in response to H1N1 IAVs. Internalization of the epidermal growth factor receptor (EGFR) was also greater in respiratory epithelial cells derived from male than female mice. IAV infection of Areg knock-out (Areg-/-) mice eliminated sex differences in IAV pathogenesis, with a more significant role for AREG in infection of male compared to female mice. Deletion of Areg had no effect on virus replication kinetics in either sex. Gonadectomy and treatment of either wild-type or Areg-/- males with testosterone improved the outcome of IAV as compared with their placebo-treated conspecifics. CONCLUSIONS: Taken together, these data show that elevated levels of testosterone and AREG, either independently or in combination, improve resilience (i.e., repair and recovery of damaged tissue) and contribute to better influenza outcomes in males compared with females.

Sex Reporting in Preclinical Microbiological and Immunological Research.

Both sex (i.e., biological construct of male and female) and gender (i.e., social construct of masculine and feminine) impact the pathogenesis of diseases, including those caused by microbial infections. Following the 2015 NIH policy for consideration of sex as a biological variable in preclinical research, in 2018, authors of papers published in primary-research American Society for Microbiology (ASM) journals will be asked to report the sex of the research subjects and animals and of materials derived directly from them. To address the need for sex reporting in ASM journals, we systematically reviewed 2,928 primary-research articles published in six primary-research ASM journals (Antimicrobial Agents and Chemotherapy, Clinical and Vaccine Immunology, Infection and Immunity, Journal of Bacteriology, Journal of Virology, and mBio) in 2016. Approximately 37% of animal studies and 9% of primary cell culture papers published in 2016 would have been affected by the new sex-reporting policy. For animal studies (i.e., studies with any nonhuman vertebrate hosts), most published papers either did not report the sex of the animals or used only female animals, and a minority used only males or both sexes. For published studies using primary cells from diverse animal species (i.e., humans and nonhuman vertebrates), almost all studies failed to report the sex of donors from which the cells were isolated. We believe that reporting the sex of animals and even of the donors of derived cells could improve the rigor and reproducibility of research conducted in microbiology and immunology and published in ASM journals.

Sex Steroids Mediate Bidirectional Interactions Between Hosts and Microbes.

The outcome of microbial infections in mammals, including humans, is affected by the age, sex, and reproductive status of the host suggesting a role for sex steroid hormones. Testosterone, estradiol, and progesterone, signaling through their respective steroid receptors, affect the functioning of immune cells to cause differential susceptibility to parasitic, bacterial, and viral infections. Microbes, including fungi, bacteria, parasites, and viruses, can also use sex steroid hormones and manipulate sex steroid receptor signaling mechanisms to increase their own survival and replication rate. The multifaceted use of sex steroid hormones by both microbes and hosts during infection forms the basis of this review. In the arms race between microbes and hosts, both hosts and microbes have evolved to utilize sex steroid hormone signaling mechanisms for survival.

Age and testosterone mediate influenza pathogenesis in male mice.

Influenza severity increases with age, with hospitalization and mortality rates during seasonal influenza epidemics being higher in older men than age-matched women. As it is known that with age, circulating testosterone levels decline in males, we hypothesized that reduced testosterone contributes to age-associated increases in influenza severity. A murine model was used to test this hypothesis. As in men, testosterone concentrations were lower in aged (18 mo) than young (2 mo) male C57BL/6 mice. Following inoculation with influenza A virus (IAV), aged males experienced greater morbidity, clinical disease, and pulmonary inflammation than young males, and had lower neutralizing and total anti-influenza IgG antibody responses. Peak titers of virus in the lungs did not differ between aged and young males, but virus clearance was delayed in aged males. In young males, removal of the gonads increased-whereas treatment of gonadectomized males with testosterone reduced-morbidity, clinical illness, and pulmonary pathology, but viral replication was not altered by hormone manipulation in young males. Treatment of aged males with testosterone improved survival following infection but did not alter either virus replication or pulmonary pathology. These results indicate that low concentrations of testosterone, whether induced surgically in young males or naturally occurring in aged males, negatively impact the outcome of influenza.

Phosphorylation of p53 by TAF1 inactivates p53-dependent transcription in the DNA damage response.

While p53 activation has long been studied, the mechanisms by which its targets genes are restored to their preactivation state are less clear. We report here that TAF1 phosphorylates p53 at Thr55, leading to dissociation of p53 from the p21 promoter and inactivation of transcription late in the DNA damage response. We further show that cellular ATP level might act as a molecular switch for Thr55 phosphorylation on the p21 promoter, indicating that TAF1 is a cellular ATP sensor. Upon DNA damage, cells undergo PARP-1-dependent ATP depletion, which is correlated with reduced TAF1 kinase activity and Thr55 phosphorylation, resulting in p21 activation. As cellular ATP levels recover, TAF1 is able to phosphorylate p53 on Thr55, which leads to dissociation of p53 from the p21 promoter. ChIP-sequencing analysis reveals p53 dissociates from promoters genome wide as cells recover from DNA damage, suggesting the general nature of this mechanism.

Peritoneal washing cytology in patients with BRCA1 or BRCA2 mutations undergoing risk-reducing salpingo-oophorectomies: a 10-year experience and reappraisal of its clinical utility.

OBJECTIVE: To evaluate the utility of peritoneal washing cytology (PWC) for detecting occult primary peritoneal carcinoma in patients with BRCA1 or BRCA2 mutations, we reviewed PWCs obtained during risk-reducing salpingo-oophorectomy (RRSO) from 117 patients at our institution and correlated the results with surgical pathology findings. METHODS: Records of 128 PWCs from 125 patients with BRCA1 or BRCA2 mutations undergoing RRSO at MD Anderson Cancer Center between 2000 and 2010 were obtained. Slides were available for review for 119 PWCs from 117 patients (2 patients had 2 PWCs each). Cytopathologists, blinded to the RRSO histopathologic diagnoses, categorized the PWCs as benign, atypical, suspicious for malignancy, or malignant. These results were correlated with the RRSO histopathologic diagnoses. RESULTS: PWCs from 113 patients were benign. Of the remaining 4 patients, 2 had PWCs classified as atypical, 1 as suspicious for malignancy, and 1 as malignant. The corresponding RRSO histopathologic findings of the 2 atypical PWCs showed endosalpingiosis and cystadenofibroma in one case and showed no abnormalities in the other case. Both patients with suspicious or malignant PWCs, indicating the possibility of occult peritoneal carcinoma, had RRSO histopathologic diagnoses of endometriosis and endosalpingiosis. Nine patients had abnormal tubal or ovarian histologic findings, but all 9 of these patients had benign PWCs. CONCLUSION: PWC has the potential to detect occult peritoneal carcinoma in patients with BRCA1 or BRCA2 mutations. The clinical significance of a positive PWC without abnormal RRSO histology remains unclear and will require long-term follow-up for determination.

p53 acetylation is crucial for its transcription-independent proapoptotic functions.

Acetylation of p53 at carboxyl-terminal lysine residues enhances its transcriptional activity associated with cell cycle arrest and apoptosis. Here we demonstrate that p53 acetylation at Lys-320/Lys-373/Lys-382 is also required for its transcription-independent functions in BAX activation, reactive oxygen species production, and apoptosis in response to the histone deacetylase inhibitors (HDACi) suberoylanilide hydroxamic acid and LAQ824. Knock-out of p53 markedly reduced HDACi-induced apoptosis. Unexpectedly, expression of transactivation-deficient p53 variants sensitized p53-null cells to HDACi-mediated BAX-dependent apoptosis, whereas knockdown of endogenous mutant p53 in cancer cells reduced HDACi-mediated cytotoxicity. Evaluation of the mechanisms controlling this response led to the discovery of a novel interaction between p53 and Ku70. The association between these two proteins was acetylation-independent, but acetylation of p53 could prevent and disrupt the Ku70-BAX complex and enhance apoptosis. These results suggest a new mechanism of acetylated p53 transcription-independent regulation of apoptosis.

An acetylation switch in p53 mediates holo-TFIID recruitment.

Posttranslational modifications mediate important regulatory functions in biology. The acetylation of the p53 transcription factor, for example, promotes transcriptional activation of target genes including p21. Here we show that the acetylation of two lysine residues in p53 promotes recruitment of the TFIID subunit TAF1 to the p21 promoter through its bromodomains. UV irradiation of cells diacetylates p53 at lysines 373 and 382, which in turn recruits TAF1 to a distal p53-binding site on the p21 promoter prior to looping to the core promoter. Disruption of acetyl-p53/bromodomain interaction inhibits TAF1 recruitment to both the distal p53-binding site and the core promoter. Further, the TFIID subunits TAF4, TAF5, and TBP are detected on the core promoter prior to TAF1, suggesting that, upon DNA damage, distinct subunits of TFIID may be recruited separately to the p21 promoter and that the transcriptional activation depends on posttranslational modification of the p53 transcription factor.

Making minimally invasive THR safe: conclusions from biomechanical simulation and analysis.

The use of smaller surgical incisions has become popularized for total hip arthroplasty (THR) because of the potential benefits of shorter recovery and improved cosmetic appearance. However, an increased incidence of serious complications has been reported. To minimize the risks of minimally invasive approaches to THR, we have developed an experimental approach which enables us to evaluate risk factors in these procedures through cadaveric simulations performed within the laboratory. During cadaveric hip replacement procedures performed via posterior and antero-lateral mini-incisions, pressures developed between the wound edges and the retractors were approximately double those recorded during conventional hip replacement using Charnley retractors (p < 0.01). In MIS procedures performed via the dual-incision approach, lack of direct visualisation of the proximal femur led to misalignment of broaches and implants with increased risk of cortical fracture during canal preparation and implant insertion. Cadaveric simulation of surgical procedures allows surgeons to measure variables affecting the technical success of surgery and to master new procedures without placing patients at risk.

A specific PP2A regulatory subunit, B56gamma, mediates DNA damage-induced dephosphorylation of p53 at Thr55.

Protein phosphatase 2A (PP2A) has been implicated to exert its tumor suppressive function via a small subset of regulatory subunits. In this study, we reported that the specific B regulatory subunits of PP2A B56gamma1 and B56gamma3 mediate dephosphorylation of p53 at Thr55. Ablation of the B56gamma protein by RNAi, which abolishes the Thr55 dephosphorylation in response to DNA damage, reduces p53 stabilization, Bax expression and cell apoptosis. To investigate the molecular mechanisms, we have shown that the endogenous B56gamma protein level and association with p53 increase after DNA damage. Finally, we demonstrate that Thr55 dephosphorylation is required for B56gamma3-mediated inhibition of cell proliferation and cell transformation. These results suggest a molecular mechanism for B56gamma-mediated tumor suppression and provide a potential route for regulation of B56gamma-specific PP2A complex function.

Disinfection using ultraviolet radiation as an antimicrobial agent: a review and synthesis of mechanisms and concerns.

Control of microbes in sensitive health care settings cannot be maintained without effective disinfection methods. Recently, increasing microbial resistance to common chemical agents, including antibiotics, has required the implementation of new approaches to disinfection where such sanitary practices are critical. Pharmaceutical manufacturers and many interrelated industries also have a need for effective disinfection and sanitation strategies. As a result, standard practices that direct and maintain effective microbial controls are established. These practices exploit a thorough understanding of biological processes and manipulate them to preserve the quality and safety of a valuable product. Most biological processes are delicately balanced with their surroundings, or environment. When changes are made to the environment during disinfection, damaged microbes may respond by repairing the immediate damage or by adapting their biological processes through developing a resistance. Unless sound disinfection procedures are consistently applied, new strains of environmental microbes that can utilize multiple resistance markers may be artificially selected. Examples of related drug resistance are currently being studied and managed in many large hospitals, but non-chemical methods are also subject to similar concerns. Here, we review the use of ultraviolet-based disinfection practices, the biological basis for them, and some potential desensitization issues that may develop. Finally, we suggest some approaches to study and practically address these effects.