The porin and the permeating antibiotic: A selective diffusion barrier in gram-negative bacteria

Gram-negative bacteria are responsible for a large proportion of antibiotic resistant bacterial diseases. These bacteria have a complex cell envelope that comprises an outer membrane and an inner membrane that delimit the periplasm. The outer membrane contains various protein channels, called porins, which are involved in the influx of various compounds, including several classes of antibiotics. Bacterial adaptation to reduce influx through porins is an increasing problem worldwide that contributes, together with efflux systems, to the emergence and dissemination of antibiotic resistance. An exciting challenge is to decipher the genetic and molecular basis of membrane impermeability as a bacterial resistance mechanism. This Review outlines the bacterial response towards antibiotic stress on altered membrane permeability and discusses recent advances in molecular approaches that are improving our knowledge of the physico-chemical parameters that govern the translocation of antibiotics through porin channel

High NEFA concentrations around parturition are associated with delayed ovulations in grazing dairy cows

The objectives of this study were to assess indicators of metabolic status of grazing dairy cows around parturition, and the relationship between these indicators with the resumption of ovulations postpartum (ROP). Holstein multiparous cows (N=20) grazing on improved pastures and supplemented with concentrates were body condition scored and tail bled weekly from wk −2 through +9 related to parturition. Plasma samples were analyzed for nonesterified fatty acids (NEFA), leptin, insulin-like growth factor-1 (IGF-1) and progesterone (P4). Data were analyzed with mixed models, logistic regression, with receiver operator characteristic (ROC), and Cox regression analysis. Cows having Delayed Ovulation ([DO], ROP on week ≥5) had lower BCS, and higher NEFAs than cows having a normal ROP around parturition (BCS: 2.73±0.08 vs. 2.94±0.05, Pb0.05, and NEFA: 0.43±0.04 vs. 0.35±0.02 mM, Pb0.10, respectively). Also, DO cows had lower BCS than normal herdmates (2.59±0.10 vs. 2.99±0.06, Pb0.01) around time of ROP, but they had similar NEFA, leptin and IGF-1. The risk for DO increased as NEFA increased (0.4% and 0.5% per every increasing mM of NEFA in prepartum and postpartum, respectively). The ROC curve showed that NEFA (prepartum and postpartum) had areas of 0.85 and 0.80, and cut-off values of 0.39 and 0.47 mM. Finally, hazard for ROP increased as prepartum IGF-1 increased, and it decreased as postpartum NEFA increased. In conclusion, cows with lower BCS and higher prepartum and postpartum NEFA had higher odds for getting DO than herdmates with greater BCS and lower NEFA concentrations.Facultad de Ciencias Veterinaria

FAST, a method based on split-GFP for the detection in solution of proteins synthesized in cell-free expression systems

Cell-free protein synthesis (CFPS) systems offer a versatile platform for a wide range of applications. However, the traditional methods for detecting proteins synthesized in CFPS, such as radioactive labeling, fluorescent tagging, or electrophoretic separation, may be impractical, due to environmental hazards, high costs, technical complexity, and time consuming procedures. These limitations underscore the need for new approaches that streamline the detection process, facilitating broader application of CFPS. By harnessing the reassembly capabilities of two GFP fragments—specifically, the GFP1-10 and GFP11 fragments—we have crafted a method that simplifies the detection of in vitro synthesized proteins called FAST (Fluorescent Assembly of Split-GFP for Translation Tests). FAST relies on the fusion of the small tag GFP11 to virtually any gene to be expressed in CFPS. The in vitro synthesized protein:GFP11 can be rapidly detected in solution upon interaction with an enhanced GFP1-10 fused to the Maltose Binding Protein (MBP:GFP1-10). This interaction produces a fluorescent signal detectable with standard fluorescence readers, thereby indicating successful protein synthesis. Furthermore, if required, detection can be coupled with the purification of the fluorescent complex using standardized MBP affinity chromatography. The method's versatility was demonstrated by fusing GFP11 to four distinct E. coli genes and analyzing the resulting protein synthesis in both a homemade and a commercial E. coli CFPS system. Our experiments confirmed that the FAST method offers a direct correlation between the fluorescent signal and the amount of synthesized protein:GFP11 fusion, achieving a sensitivity threshold of 8 ± 2 pmol of polypeptide, with fluorescence plateauing after 4 h. Additionally, FAST enables the investigation of translation inhibition by antibiotics in a dose-dependent manner. In conclusion, FAST is a new method that permits the rapid, efficient, and non-hazardous detection of protein synthesized within CFPS systems and, at the same time, the purification of the target protein

Metritis in dairy cows: Risk factors and reproductive performance

The objectives of this study were to assess the risk factors for metritis, its effects on milk yield and on reproductive performance, and the efficacy of ceftiofur therapy in Holstein dairy cows. Cows (n. =. 303) from a commercial dairy herd in Argentina were studied. Cows were scored for body condition, and blood samples were collected on d -14, 7, 21, 31, 41, and 50 relative to parturition. Cows having a watery, purulent, or brown, and fetid vaginal discharge (VD) and rectal temperature ≤39.2°C were diagnosed as having clinical metritis, and those having a similar VD and rectal temperature >39.2°C were diagnosed as having puerperal metritis. Both clinical and puerperal metritis cows were randomly assigned to control (no treatment) or ceftiofur group (2.2. mg/kg. ×. 3 consecutive days). Cure was declared if clear VD was observed at 21 d in milk (DIM). Blood samples were analyzed for nonesterified fatty acids, β-hydroxybutyrate, and blood urea nitrogen using commercial kits, and for insulin-like growth factor-1, insulin, and leptin by RIA. Data were analyzed with PROC MIXED, GENMOD, PHREG, and LIFETEST from SAS (SAS Institute Inc., Cary, NC). The risk for metritis increased with dystocia, retained fetal membranes, and dead calf [AOR (adjusted odds ratio). =. 2.58, 95% CI: 1.189-5.559], and as prepartum nonesterified fatty acids levels increased (AOR. =. 1.001, 95% CI: 0.999-1.002). Conversely, risk decreased as prepartum insulin-like growth factor-1 increased (AOR. =. 0.65, 95% CI: 0.349-1.219). Cows having either clinical or puerperal metritis produced less milk by 90 DIM than did healthy cows (2,236. ±. 172 vs. 2,367. ±. 77 vs. 2,647. ±. 82 kg, respectively). Cows with puerperal metritis had lower risk for pregnancy by 100 DIM (AOR. =. 0.189, 95% CI: 0.070-0.479) and a lower hazard rate for pregnancy by 150 DIM (hazard rate: 0.753, 95% CI: 0.621-0.911), and took longer to get pregnant (129 vs. 111 vs. 109 d, for puerperal metritis, clinical metritis, and healthy cows, respectively). Ceftiofur treatment was not associated with cure rate or milk yield but was related to increased risk for pregnancy at timed artificial insemination (AOR. =. 2.688, 95% CI: 0.687-10.832), and for lower risk of reproductive cull (AOR. =. 0.121, 95% CI: 0.014-1.066). In conclusion, abnormal calving and negative energy balance are associated with increased risk for metritis. Metritis, especially puerperal metritis, correlates with reduced milk production and poor reproductive performance. Finally, the likelihood for having a normal VD (indicative of cure) increased 2.6% for every day of increase in postpartum time and was 2 times higher for cows with clinical metritis than for those with puerperal metritis.Facultad de Ciencias Veterinaria

The translation inhibitors kasugamycin, edeine and GE81112 target distinct steps during 30S initiation complex formation

During bacterial translation initiation, the 30S ribosomal subunit, initiation factors, and initiator tRNA define the reading frame of the mRNA. This process is inhibited by kasugamycin, edeine and GE81112, however, their mechanisms of action have not been fully elucidated. Here we present cryo-electron microscopy structures of 30S initiation intermediate complexes formed in the presence of kasugamycin, edeine and GE81112 at resolutions of 2.0-2.9 & Aring;. The structures reveal that all three antibiotics bind within the E-site of the 30S and preclude 30S initiation complex formation. While kasugamycin and edeine affect early steps of 30S pre-initiation complex formation, GE81112 stalls pre-initiation complex formation at a further step by allowing start codon recognition, but impeding IF3 departure. Collectively, our work highlights how chemically distinct compounds binding at a conserved site on the 30S can interfere with translation initiation in a unique manner

Small-Group Learning in an Upper-Level University Biology Class Enhances Academic Performance and Student Attitudes Toward Group Work

To improve science learning, science educators' teaching tools need to address two major criteria: teaching practice should mirror our current understanding of the learning process; and science teaching should reflect scientific practice. We designed a small-group learning (SGL) model for a fourth year university neurobiology course using these criteria and studied student achievement and attitude in five course sections encompassing the transition from individual work-based to SGL course design. All students completed daily quizzes/assignments involving analysis of scientific data and the development of scientific models. Students in individual work-based (Individualistic) sections usually worked independently on these assignments, whereas SGL students completed assignments in permanent groups of six. SGL students had significantly higher final exam grades than Individualistic students. The transition to the SGL model was marked by a notable increase in 10th percentile exam grade (Individualistic: 47.5%; Initial SGL: 60%; Refined SGL: 65%), suggesting SGL enhanced achievement among the least prepared students. We also studied student achievement on paired quizzes: quizzes were first completed individually and submitted, and then completed as a group and submitted. The group quiz grade was higher than the individual quiz grade of the highest achiever in each group over the term. All students – even term high achievers –could benefit from the SGL environment. Additionally, entrance and exit surveys demonstrated student attitudes toward SGL were more positive at the end of the Refined SGL course. We assert that SGL is uniquely-positioned to promote effective learning in the science classroom

High NEFA concentrations around parturition are associated with delayed ovulations in grazing dairy cows

The objectives of this study were to assess indicators of metabolic status of grazing dairy cows around parturition, and the relationship between these indicators with the resumption of ovulations postpartum (ROP). Holstein multiparous cows (N=20) grazing on improved pastures and supplemented with concentrates were body condition scored and tail bled weekly from wk −2 through +9 related to parturition. Plasma samples were analyzed for nonesterified fatty acids (NEFA), leptin, insulin-like growth factor-1 (IGF-1) and progesterone (P4). Data were analyzed with mixed models, logistic regression, with receiver operator characteristic (ROC), and Cox regression analysis. Cows having Delayed Ovulation ([DO], ROP on week ≥5) had lower BCS, and higher NEFAs than cows having a normal ROP around parturition (BCS: 2.73±0.08 vs. 2.94±0.05, Pb0.05, and NEFA: 0.43±0.04 vs. 0.35±0.02 mM, Pb0.10, respectively). Also, DO cows had lower BCS than normal herdmates (2.59±0.10 vs. 2.99±0.06, Pb0.01) around time of ROP, but they had similar NEFA, leptin and IGF-1. The risk for DO increased as NEFA increased (0.4% and 0.5% per every increasing mM of NEFA in prepartum and postpartum, respectively). The ROC curve showed that NEFA (prepartum and postpartum) had areas of 0.85 and 0.80, and cut-off values of 0.39 and 0.47 mM. Finally, hazard for ROP increased as prepartum IGF-1 increased, and it decreased as postpartum NEFA increased. In conclusion, cows with lower BCS and higher prepartum and postpartum NEFA had higher odds for getting DO than herdmates with greater BCS and lower NEFA concentrations.Facultad de Ciencias Veterinaria

Price Effects of Sovereign Debt Auctions in the Euro-Zone: The Role of the Crisis

Exploring the period since the inception of the euro, we show that secondary-market yields on Italian public debt increase in anticipation of auctions of new issues and decrease after the auction, while no or a smaller such effect is present for German public debt. However, these yield movements on the Italian debt are largely confined to the period of the crisis since mid-2007. We also find that there is some tendency of the yield movements to be larger when the demand for the new issue is smaller relative to its supply. Our results are consistent with a framework in which a small group of primary dealers require compensation for inventory risk and this compensation needs to be higher when market uncertainty is larger. We also find that the secondary-market behaviour of series with a maturity close to the auctioned series, but for which there is no auction, is very similar to the secondary-market behaviour of the auctioned series. These findings support an explanation of yield movements based on the behaviour of primary dealers with limited risk-bearing capacity

Metritis in dairy cows: Risk factors and reproductive performance

The objectives of this study were to assess the risk factors for metritis, its effects on milk yield and on reproductive performance, and the efficacy of ceftiofur therapy in Holstein dairy cows. Cows (n. =. 303) from a commercial dairy herd in Argentina were studied. Cows were scored for body condition, and blood samples were collected on d -14, 7, 21, 31, 41, and 50 relative to parturition. Cows having a watery, purulent, or brown, and fetid vaginal discharge (VD) and rectal temperature ≤39.2°C were diagnosed as having clinical metritis, and those having a similar VD and rectal temperature >39.2°C were diagnosed as having puerperal metritis. Both clinical and puerperal metritis cows were randomly assigned to control (no treatment) or ceftiofur group (2.2. mg/kg. ×. 3 consecutive days). Cure was declared if clear VD was observed at 21 d in milk (DIM). Blood samples were analyzed for nonesterified fatty acids, β-hydroxybutyrate, and blood urea nitrogen using commercial kits, and for insulin-like growth factor-1, insulin, and leptin by RIA. Data were analyzed with PROC MIXED, GENMOD, PHREG, and LIFETEST from SAS (SAS Institute Inc., Cary, NC). The risk for metritis increased with dystocia, retained fetal membranes, and dead calf [AOR (adjusted odds ratio). =. 2.58, 95% CI: 1.189-5.559], and as prepartum nonesterified fatty acids levels increased (AOR. =. 1.001, 95% CI: 0.999-1.002). Conversely, risk decreased as prepartum insulin-like growth factor-1 increased (AOR. =. 0.65, 95% CI: 0.349-1.219). Cows having either clinical or puerperal metritis produced less milk by 90 DIM than did healthy cows (2,236. ±. 172 vs. 2,367. ±. 77 vs. 2,647. ±. 82 kg, respectively). Cows with puerperal metritis had lower risk for pregnancy by 100 DIM (AOR. =. 0.189, 95% CI: 0.070-0.479) and a lower hazard rate for pregnancy by 150 DIM (hazard rate: 0.753, 95% CI: 0.621-0.911), and took longer to get pregnant (129 vs. 111 vs. 109 d, for puerperal metritis, clinical metritis, and healthy cows, respectively). Ceftiofur treatment was not associated with cure rate or milk yield but was related to increased risk for pregnancy at timed artificial insemination (AOR. =. 2.688, 95% CI: 0.687-10.832), and for lower risk of reproductive cull (AOR. =. 0.121, 95% CI: 0.014-1.066). In conclusion, abnormal calving and negative energy balance are associated with increased risk for metritis. Metritis, especially puerperal metritis, correlates with reduced milk production and poor reproductive performance. Finally, the likelihood for having a normal VD (indicative of cure) increased 2.6% for every day of increase in postpartum time and was 2 times higher for cows with clinical metritis than for those with puerperal metritis.Facultad de Ciencias Veterinaria

Rfam: Wikipedia, clans and the “decimal” release

The Rfam database aims to catalogue non-coding RNAs through the use of sequence alignments and statistical profile models known as covariance models. In this contribution, we discuss the pros and cons of using the online encyclopedia, Wikipedia, as a source of community‐derived annotation. We discuss the addition of groupings of related RNA families into clans and new developments to the website. Rfam is available on the Web at http://rfam.sanger.ac.uk