DrwH, a novel WHy domain-containing hydrophobic LEA5C protein from Deinococcus radiodurans, protects enzymatic activity under oxidative stress.

Water stress and hypersensitive response (WHy) domain is typically found as a component of atypical late embryogenesis abundant (LEA) proteins closely associated with resistance to multiple stresses in numerous organisms. Several putative LEA proteins have been identified in Deinococcus bacteria; however their precise function remains unclear. This work reports the characterization of a Deinococcus-specific gene encoding a novel WHy domain-containing hydrophobic LEA5C protein (named DrwH) in D. radiodurans R1. The expression of the drwH gene was induced by oxidative and salinity stresses. Inactivation of this gene resulted in increased sensitivity to oxidative and salinity stresses as well as reduced activities of antioxidant enzymes. The WHy domain of the DrwH protein differs structurally from that of a previously studied bacterial LEA5C protein, dWHy1, identified as a gene product from an Antarctic desert soil metagenome library. Further analysis indicated that in E. coli, the function of DrwH is related to oxidative stress tolerance, whereas dWHy1 is associated with freezing-thawing stress tolerance. Under oxidative stress induced by H2O2, DrwH protected the enzymatic activities of malate dehydrogenase (MDH) and lactate dehydrogenase (LDH). These findings provide new insight into the evolutionary and survival strategies of Deinococcus bacteria under extreme environmental conditions.

An enhanced vector-free allele exchange (VFAE) mutagenesis protocol for genome editing in a wide range of bacterial species.

Vector-free allele exchange (VFAE) is a newly developed protocol for genome editing in Pseudomonas species. Although several parameters have been determined to optimize the procedures for obtaining a stable and high-frequency mutation, numerous false-positive clones still appear on the plate, which increases the difficulty of finding the desired mutants. It has also not been established whether this protocol can be used for genome editing in other bacterial species. In the current study, the protocol was modified to dramatically decrease the occurrence of false-positive colonies using Pseudomonas stutzeri A1501 as a model strain. This improvement was reached by increasing the occurrence of circular-DNA cassettes of the correct size. Furthermore, the enhanced protocol was used to construct mutants in both the gram-negative Escherichia coli BL21 and gram-positive Bacillus subtilis 168 strains. The protocol works well in both strains, yielding ideal results with a low percentage of false-positive colonies. In summary, the enhanced VFAE mutagenesis protocol is a potential tool for use in bacterial genome editing.

High-Frequency Targeted Mutagenesis in Pseudomonas stutzeri Using a Vector-Free Allele-Exchange Protocol.

The complexity of the bacterial recombination system is a barrier for the construction of bacterial mutants for the further functional investigation of specific genes. Several protocols have been developed to inactivate genes from the genus Pseudomonas. Those protocols are complicated and time-consuming and mostly do not enable easy construction of multiple knock-ins/outs. The current study describes a single and double crossover-recombination system using an optimized vector-free allele-exchange protocol for gene disruption and gene replacement in a single species of the family Pseudomonadaceae. The protocol is based on self-ligation (circularization) for the DNA cassette which has been obtained by overlapping polymerase chain reaction (Fusion-PCR), and carries an antibiotic resistance cassette flanked by homologous internal regions of the target locus. To establish the reproducibility of the approach, three different chromosomal genes (ncRNA31, rpoN, rpoS) were knocked-out from the root-associative bacterium Pseudomonas stutzeri A1501. The results showed that the P. stutzeri A1501 mutants, which are free of any plasmid backbone, could be obtained via a single or double crossover recombination. In order to optimize this protocol, three key factors that were found to have great effect on the efficiency of the homologous recombination were further investigated. Moreover, the modified protocol does not require further cloning steps, and it enables the construction of multiple gene knock-in/out mutants sequentially. This work provides a simple and rapid mutagenesis strategy for genome editing in P. stutzeri, which may also be applicable for other gram-negative bacteria.

Occupational traumatic injuries among workers in health care facilities - United States, 2012-2014.

In 2013, one in five reported nonfatal occupational injuries occurred among workers in the health care and social assistance industry, the highest number of such injuries reported for all private industries. In 2011, U.S. health care personnel experienced seven times the national rate of musculoskeletal disorders compared with all other private sector workers. To reduce the number of preventable injuries among health care personnel, CDC's National Institute for Occupational Safety and Health (NIOSH), with collaborating partners, created the Occupational Health Safety Network (OHSN) to collect detailed injury data to help target prevention efforts. OHSN, a free, voluntary surveillance system for health care facilities, enables prompt and secure tracking of occupational injuries by type, occupation, location, and risk factors. This report describes OHSN and reports on current findings for three types of injuries. A total of 112 U.S. facilities reported 10,680 OSHA-recordable* patient handling and movement (4,674 injuries); slips, trips, and falls (3,972 injuries); and workplace violence (2,034 injuries) injuries occurring from January 1, 2012-September 30, 2014. Incidence rates for patient handling; slips, trips, and falls; and workplace violence were 11.3, 9.6, and 4.9 incidents per 10,000 worker-months,† respectively. Nurse assistants and nurses had the highest injury rates of all occupations examined. Focused interventions could mitigate some injuries. Data analyzed through OHSN identify where resources, such as lifting equipment and training, can be directed to potentially reduce patient handling injuries. Using OHSN can guide institutional and national interventions to protect health care personnel from common, disabling, preventable injuries.

Physiological studies on microalgal culture additives to optimize growth rate and oil content.

Insulin, in nature, has a stimulatory effect on microorganisms. These effects include the acceleration of sugar metabolism, triacylglycerol anabolism, growth rate, and formation of oils. We also observed that insulin may cause indirect activation of triacylglycerol lipase by forcing the cell to permanently require an energy source. Thus, cells can consume all of their accumulated internal fuel sources such as lipids, proteins, and carbohydrates. After studying the effects of using two types of insulin (Humulin 70/30, and human insulin expressed in yeast) at different concentrations on microalgae (Chlorella sp.), we found that with certain concentrations of insulin (1:3.3 ml unit Humulin 70/30 per ml; 1:2.6 ml unit yeast insulin per ml), there was an increase in algal growth rate and decrease in cell size. We therefore studied the effect of insulin under conditions of lipase inhibition by Triton WR 1339 (Tyloxapol), which was used at different concentrations with and without insulin. We found strong regression in the growth rate with increasing Triton concentrations. However, we also observed that the cell size under the effect of Triton and Triton-insulin was larger than the cell size under the effect of insulin alone, and also larger than for control cells. Also, the oil content of the Triton-insulin cells was higher than those of the control cells or the cells under the effect of insulin alone.

Increase in sharps injuries in surgical settings versus nonsurgical settings after passage of national needlestick legislation.

BACKGROUND: The operating room is a high-risk setting for occupational sharps injuries and bloodborne pathogen exposure. The requirement to provide safety-engineered devices, mandated by the Needlestick Safety and Prevention Act of 2000, has received scant attention in surgical settings. STUDY DESIGN: We analyzed percutaneous injury surveillance data from 87 hospitals in the United States from 1993 through 2006, comparing injury rates in surgical and nonsurgical settings before and after passage of the law. We identified devices and circumstances associated with injuries among surgical team members. RESULTS: Of 31,324 total sharps injuries, 7,186 were to surgical personnel. After the legislation, injury rates in nonsurgical settings dropped 31.6%, but increased 6.5% in surgical settings. Most injuries were caused by suture needles (43.4%), scalpel blades (17%), and syringes (12%). Three-quarters of injuries occurred during use or passing of devices. Surgeons and residents were most often original users of the injury-causing devices; nurses and surgical technicians were typically injured by devices originally used by others. CONCLUSIONS: Despite legislation and advances in sharps safety technology, surgical injuries continued to increase during the period that nonsurgical injuries decreased significantly. Hospitals should comply with requirements for the adoption of safer surgical technologies, and promote policies and practices shown to substantially reduce blood exposures to surgeons, their coworkers, and patients. Although decisions affecting the safety of the surgical team lie primarily in the surgeon's hands, there are also roles for administrators, educators, and policy makers.

Increase in sharps injuries in surgical settings versus nonsurgical settings after passage of national needlestick legislation.

BACKGROUND: The operating room is a high-risk setting for occupational sharps injuries and bloodborne pathogen exposure. The requirement to provide safety-engineered devices, mandated by the Needlestick Safety and Prevention Act of 2000, has received scant attention in surgical settings. STUDY DESIGN: We analyzed percutaneous injury surveillance data from 87 hospitals in the United States from 1993 through 2006, comparing injury rates in surgical and nonsurgical settings before and after passage of the law. We identified devices and circumstances associated with injuries among surgical team members. RESULTS: Of 31,324 total sharps injuries, 7,186 were to surgical personnel. After the legislation, injury rates in nonsurgical settings dropped 31.6%, but increased 6.5% in surgical settings. Most injuries were caused by suture needles (43.4%), scalpel blades (17%), and syringes (12%). Three-quarters of injuries occurred during use or passing of devices. Surgeons and residents were most often original users of the injury-causing devices; nurses and surgical technicians were typically injured by devices originally used by others. CONCLUSIONS: Despite legislation and advances in sharps safety technology, surgical injuries continued to increase during the period that nonsurgical injuries decreased significantly. Hospitals should comply with requirements for the adoption of safer surgical technologies, and promote policies and practices shown to substantially reduce blood exposures to surgeons, their coworkers, and patients. Although decisions affecting the safety of the surgical team lie primarily in the surgeon's hands, there are also roles for administrators, educators, and policy makers.