Impacts of Upstream Drought and Water Withdrawals on the Health and Survival of Downstream Estuarine Oyster Populations

Increases in the frequency, duration, and severity of regional drought pose major threats to the health and integrity of downstream ecosystems. During 2007-2008, the U.S. southeast experienced one of the most severe droughts on record. Drought and water withdrawals in the upstream watershed led to decreased freshwater input to Apalachicola Bay, Florida, an estuary that is home to a diversity of commercially and ecologically important organisms. This study applied a combination of laboratory experiments and field observations to investigate the effects of reduced freshwater input on Apalachicola oysters. Oysters suffered significant disease-related mortality under high-salinity, drought conditions, particularly during the warm summer months. Mortality was size-specific, with large oysters of commercially harvestable size being more susceptible than small oysters. A potential salinity threshold was revealed between 17 and 25 ppt, where small oysters began to suffer mortality, and large oysters exhibited an increase in mortality. These findings have important implications for watershed management, because upstream freshwater releases could be carefully timed and allocated during stressful periods of the summer to reduce disease-related oyster mortality. Integrated, forward-looking water management is needed, particularly under future scenarios of climate change and human population growth, to sustain the valuable ecosystem services on which humans depend

Antibody responses to a Cryptosporidium parvum rCP15/60 vaccine

Cryptosporidium parvum is a zoonotic apicomplexa-protozoan pathogen that causes gastroenteritis and diarrhoea in mammals worldwide. The organism is transmitted by ingestion of oocysts, which are shed in faeces, and completes its lifecycle in a single host.^1^ C. parvum is ubiquitous on dairy operations worldwide and is one of the leading causes of diarrhoea in calves on these farms.^2,3^ Here, for the first time, we describe the antibody response in a large group of cows to a recombinant C. parvum oocyst surface protein (rCP15/60) vaccine and the antibody response in calves fed rCP15/60-immune colostrum produced by these vaccinated cows. Results of recent genotype surveys indicate that calves are the only major reservoir for C. parvum infections in humans.^4^ Human C. parvum infections are particularly prevalent and often fatal in neonates in developing countries and to immunocompromised people, such as AIDs patients.^4^ Drug therapy against cryptosporidiosis is limited and not wholly efficacious in either humans or calves^5^, making development of an effective vaccine of paramount importance. To date, there is no commercially available effective vaccine against C. parvum, although passive immunization utilizing different zoite surface (glyco)proteins has showed promise.^6-9^ All cows we vaccinated produced an antibody response to the rCP15/60 vaccine and the magnitude of response correlated strongly with the subsequent level of antibody in their colostrum. All calves fed rCP15/60-immune colostrum showed a dose-dependent absorption of antibody. Our results demonstrate that vaccination of cows with rCP15/60 successfully induces antibodies against CP15/60 in their serum and colostrum and that these antibodies are then well absorbed when fed to neonatal calves. With further research, this C. parvum vaccine may well be a practical method of conferring passive protection to calves against cryptosporidiosis. Furthermore, a specifically targeted immune-colostrum may be valuable in protection and treatment of immunocompromised human patients with cryptosporidiosis

Decreased progesterone receptor isoform expression in luteal phase fallopian tube epithelium and high-grade serous carcinoma

We previously reported that BRCA1/2-mutated fallopian tube epithelium (FTE) collected during the luteal phase exhibits gene expression profiles more closely resembling that of high-grade serous carcinoma (HGSC) specimens than FTE collected during the follicular phase or from control patients. Since the luteal phase is characterised by high levels of progesterone, we determined whether the expression of progesterone receptor (PR) and PR-responsive genes was altered in FTE obtained from BRCA mutation carriers during the luteal phase of the menstrual cycle. RT-qPCR confirmed a decreased expression of PR mRNA in FTE during the luteal phase relative to follicular phase, in both BRCA1/2 mutation carriers and control patients. Immunohistochemistry using isoform-specific antibodies confirmed a low level of both PR-A and PR-B in HGSC and a lower level of staining in FTE samples obtained during the luteal phase compared with the follicular phase. No significant difference in PR-A or PR-B staining was found based on patient BRCA mutation status. Analysis of our previously reported gene expression profiles based upon known PR-A- and PR-B-specific target genes did not partition samples by BRCA mutation status, indicating that overall FTE PR response is not altered in BRCA mutation carriers. HGSC samples grouped separately from other samples, consistent with the observed loss of PR expression. These findings indicate no overall difference in PR signalling in FTE as a function of BRCA mutation status. Thus, the molecular similarity of BRCA1/2-mutated luteal phase FTE and HGSC likely results from an altered response to luteal phase factors other than progesterone

Design of University Small-Scale Dairy Processing Facility

This project was assigned to research the feasibility and value of implementing a dairy processing facility on the campus of the University of Nebraska-Lincoln. The facility would process milk produced from cows under the same roof and will serve as an educational experience for Nebraska dairy farmers, UNL students, and K-12 students in the Lincoln-Lancaster County area. If the project is successful and replicated across the state, this facility could have a significant impact on the reduction of milk transportation costs in the Nebraska dairy industry. The project began with researching milk processing methods and steps from production to consumption. Shortly after this step, information on milk consumption patterns was collected from UNL Dining Services to determine demand on campus. Every unit operation requires certain equipment to effectively ensure the safety and quality of the final product, and mass balances from UNL milk consumption data were used to size equipment and storage capacity. Engineering firms were then consulted to gather information on equipment specifications and prices. Equipment costs and operating costs (estimated with the help of Dr. Howell and other university dairy operations) were entered into a Monte Carlo simulation to analyze return on investment and a breakeven point. The results from the costs section showed that the fixed costs (equipment and engineering) for the milk processing would be about $1.2 million. The Monte Carlo simulation showed that the project would not turn a profit for 10-12 years, and approximately 2.25 million gallons of milk would need to be processed and sold to recover initial costs. Overall, the project successfully displays data that can be interpreted by the client to decide whether to move forward with the project and the appropriate scale for the project at UNL

Advanced Glycation End Products Acutely Impair Ca2+ Signaling in Bovine Aortic Endothelial Cells

Post-translational modification of proteins in diabetes, including formation of advanced glycation end products (AGEs) are believed to contribute to vascular dysfunction and disease. Impaired function of the endothelium is an early indicator of vascular dysfunction in diabetes and as many endothelial cell processes are dependent upon intracellular [Ca2+] and Ca2+ signalling, the aim of this study was to examine the acute effects of AGEs on Ca2+ signalling in bovine aortic endothelial cells (BAEC). Ca2+ signalling was studied using the fluorescent indicator dye Fura2-AM. AGEs were generated by incubating bovine serum albumin with 0 - 250 mM glucose or glucose-6-phosphate for 0 to 120 days at 37ºC. Under all conditions, the main AGE species generated was carboxymethyl lysine (CML) as assayed using both GC-MS and HPLC. In Ca2+-replete solution, exposure of BAEC to AGEs for 5 min caused an elevation in basal [Ca2+] and attenuated the increase in intracellular [Ca2+] caused by ATP (100 µM). In the absence of extracellular Ca2+, exposure of BAEC to AGEs for 5 min caused an elevation in basal [Ca2+] and attenuated subsequent intracellular Ca2+ release caused by ATP, thapsigargin (0.1 µM) and ionomycin (3 µM), but AGEs did not affect extracellular Ca2+ entry induced by the re-addition of Ca2+ to the bathing solution in the presence of any of these agents. The anti-oxidant α-lipoic acid (2 µM) and NAD(P)H oxidase inhibitors apocynin (500 µM) and diphenyleneiodonium (DPI, 1 µM) abolished these effects of AGEs on BAECs, as did the IP3 receptor antagonist xestospongin C (1 µM). In summary, AGEs caused an acute depletion of Ca2+ from the intracellular store in BAECs, such that the Ca2+ signal stimulated by the subsequent application other agents acting upon this store is reduced. The mechanism may involve generation of ROS from NAD(P)H oxidase and possible activation of the IP3 receptor

An exploration of grip force regulation with a low-impedance myoelectric prosthesis featuring referred haptic feedback

Abstract Background Haptic display technologies are well suited to relay proprioceptive, force, and contact cues from a prosthetic terminal device back to the residual limb and thereby reduce reliance on visual feedback. The ease with which an amputee interprets these haptic cues, however, likely depends on whether their dynamic signal behavior corresponds to expected behaviors—behaviors consonant with a natural limb coupled to the environment. A highly geared motor in a terminal device along with the associated high back-drive impedance influences dynamic interactions with the environment, creating effects not encountered with a natural limb. Here we explore grasp and lift performance with a backdrivable (low backdrive impedance) terminal device placed under proportional myoelectric position control that features referred haptic feedback. Methods We fabricated a back-drivable terminal device that could be used by amputees and non-amputees alike and drove aperture (or grip force, when a stiff object was in its grasp) in proportion to a myoelectric signal drawn from a single muscle site in the forearm. In randomly ordered trials, we assessed the performance of N=10 participants (7 non-amputee, 3 amputee) attempting to grasp and lift an object using the terminal device under three feedback conditions (no feedback, vibrotactile feedback, and joint torque feedback), and two object weights that were indiscernible by vision. Results Both non-amputee and amputee participants scaled their grip force according to the object weight. Our results showed only minor differences in grip force, grip/load force coordination, and slip as a function of sensory feedback condition, though the grip force at the point of lift-off for the heavier object was significantly greater for amputee participants in the presence of joint torque feedback. An examination of grip/load force phase plots revealed that our amputee participants used larger safety margins and demonstrated less coordination than our non-amputee participants. Conclusions Our results suggest that a backdrivable terminal device may hold advantages over non-backdrivable devices by allowing grip/load force coordination consistent with behaviors observed in the natural limb. Likewise, the inconclusive effect of referred haptic feedback on grasp and lift performance suggests the need for additional testing that includes adequate training for participants.http://deepblue.lib.umich.edu/bitstream/2027.42/116041/1/12984_2015_Article_98.pd

Investigation of translocation, DNA unwinding, and protein displacement by NS3h, the helicase domain from the Hepatitis C virus helicase

Helicases are motor proteins that are involved in DNA and RNA metabolism, replication, recombination, transcription and repair. The motors are powered by ATP binding and hydrolysis. Hepatitis C virus encodes a helicase called non-structural protein (NS3). NS3 possesses protease and helicase activities on its N-terminal and C-terminal domains respectively. The helicase domain of NS3 protein is referred as NS3h. In vitro, NS3h catalyzes RNA and DNA unwinding in a 3’ to -5’ direction. The directionality for unwinding is thought to arise in part from the enzyme's ability to translocate along DNA, but translocation has not been shown explicitly. We examined the DNA translocase activity of NS3h by using single-stranded oligonucleotide substrates containing a fluorescent probe on the 5’ end. NS3h can bind to the ssDNA and in the presence of ATP, move towards the 5’-end. When the enzyme encounters the fluorescent probe, a fluorescence change is observed that allows translocation to be characterized. Under conditions that favor binding of one NS3h per DNA substrate (100 nM NS3h, 200 nM oligonucleotide) we find that NS3h translocates on ssDNA at a rate of 46 ± 5 nt s−1 and that it can move for 230 ± 60 nt before dissociating from the DNA. The translocase activity of some helicases is responsible for displacing proteins that are bound to DNA. We studied protein displacement by using a ssDNA oligonucleotide covalently linked to biotin on the 5’-end. Upon addition of streptavidin, a ‘protein-block’ was placed in the pathway of the helicase. Interestingly, NS3h was unable to displace streptavidin from the end of the oligonucleotide, despite its ability to translocate along the DNA. The DNA unwinding activity of NS3h was examined using a 22 bp duplex DNA substrate under conditions that were identical to those used to study translocation. NS3h exhibited little or no DNA unwinding under single cycle conditions, supporting the conclusion that NS3h is a relatively poor helicase in its monomeric form, as has been reported. In summary, NS3h translocates on ssDNA as a monomer, but the translocase activity does not correspond to comparable DNA unwinding activity or protein-displacement activity under identical conditions