Gas Tunnel Engineering of Prolyl Hydroxylase Reprograms Hypoxia Signaling in Cells.

Cells have evolved intricate mechanisms for recognizing and responding to changes in oxygen (O2) concentrations. Here, we have reprogrammed cellular hypoxia (low O2) signaling via gas tunnel engineering of prolyl hydroxylase 2 (PHD2), a non-heme iron dependent O2 sensor. Using computational modeling and protein engineering techniques, we identify a gas tunnel and critical residues therein that limit the flow of O2 to PHD2's catalytic core. We show that systematic modification of these residues can open the constriction topology of PHD2's gas tunnel. Using kinetic stopped-flow measurements with NO as a surrogate diatomic gas, we demonstrate up to 3.5-fold enhancement in its association rate to the iron center of tunnel-engineered mutants. Our most effectively designed mutant displays 9-fold enhanced catalytic efficiency (kcat/KM=830±40 M-1 s-1) in hydroxylating a peptide mimic of hypoxia inducible transcription factor HIF-1α, as compared to WT PHD2 (kcat/KM=90±9 M-1 s-1). Furthermore, transfection of plasmids that express designed PHD2 mutants in HEK-293T mammalian cells reveal significant reduction of HIF-1α and downstream hypoxia response transcripts under hypoxic conditions of 1 % O2. Overall, these studies highlight activation of PHD2 as a new pathway to reprogram hypoxia responses and HIF signaling in cells.

Understanding ATP Binding to DosS Catalytic Domain with a Short ATP-Lid.

DosS is a heme-containing histidine kinase that triggers dormancy transformation inMycobacterium tuberculosis. Sequence comparison of the catalytic ATP-binding (CA) domain of DosS to other well-studied histidine kinases reveals a short ATP-lid. This feature has been thought to block binding of ATP to DosS's CA domain in the absence of interactions with DosS's dimerization and histidine phospho-transfer (DHp) domain. Here, we use a combination of computational modeling, structural biology, and biophysical studies to re-examine ATP-binding modalities in DosS. We show that the closed-lid conformation observed in crystal structures of DosS CA is caused by the presence of Zn2+ in the ATP binding pocket that coordinates with Glu537 on the ATP-lid. Furthermore, circular dichroism studies and comparisons of DosS CA's crystal structure with its AlphaFold model and homologous DesK reveal that residues 503-507 that appear as a random coil in the Zn2+-coordinated crystal structure are in fact part of the N-box α helix needed for efficient ATP binding. Such random-coil transformation of an N-box α helix turn and the closed-lid conformation are both artifacts arising from large millimolar Zn2+ concentrations used in DosS CA crystallization buffers. In contrast, in the absence of Zn2+, the short ATP-lid of DosS CA has significant conformational flexibility and can effectively bind AMP-PNP (Kd = 53 ± 13 µM), a non-hydrolyzable ATP analog. Furthermore, the nucleotide affinity remains unchanged when CA is conjugated to the DHp domain (Kd = 51 ± 6 µM). In all, our findings reveal that the short ATP-lid of DosS CA does not hinder ATP binding and provide insights that extend to 2988 homologous bacterial proteins containing such ATP-lids.

Alleles of unc-33/CRMP exhibit defects during Caenorhabditis elegans epidermal morphogenesis.

BACKGROUND: Microtubule-associated proteins regulate the dynamics, organization, and function of microtubules, impacting a number of vital cellular processes. CRMPs have been shown to control microtubule assembly and axon outgrowth during neuronal differentiation. While many microtubule-associated proteins have been linked to roles in cell division and neuronal development, it is still unclear the complement that control the formation of parallel microtubule arrays in epithelial cells. RESULTS: Here we show through time-lapse DIC microscopy that Caenorhabditis elegans embryos homozygous for the weak loss-of-function allele unc-33(e204) progress more slowly through epidermal morphogenesis, while animals homozygous for strong loss-of-function alleles exhibit more embryonic lethality. Identification of two novel missense mutations in unc-33(e572), Val476Gly, and Ser731Thr, lead to computational approaches to determine the potential effects of these changes on UNC-33/CRMP structure. Molecular dynamics simulations show that for Asp389Asn and Arg502His, two other known missense mutations, local changes in protein-protein hydrogen bonding affect the stability of the protein. However, the Val476Gly/Ser731Thr combination does not alter the structure or energetics of UNC-33 drastically when compared to the wild-type protein. CONCLUSIONS: These results support a novel role for UNC-33/CRMP in C. elegans epidermal development and shed light on how individual amino acid changes cause a loss-of-function in UNC-33.

Biflavonoid-Induced Disruption of Hydrogen Bonds Leads to Amyloid-ß Disaggregation.

Deposition of amyloid ß (Aß) fibrils in the brain is a key pathologic hallmark of Alzheimer's disease. A class of polyphenolic biflavonoids is known to have anti-amyloidogenic effects by inhibiting aggregation of Aß and promoting disaggregation of Aß fibrils. In the present study, we further sought to investigate the structural basis of the Aß disaggregating activity of biflavonoids and their interactions at the atomic level. A thioflavin T (ThT) fluorescence assay revealed that amentoflavone-type biflavonoids promote disaggregation of Aß fibrils with varying potency due to specific structural differences. The computational analysis herein provides the first atomistic details for the mechanism of Aß disaggregation by biflavonoids. Molecular docking analysis showed that biflavonoids preferentially bind to the aromatic-rich, partially ordered N-termini of Aß fibril via the π-π interactions. Moreover, docking scores correlate well with the ThT EC50 values. Molecular dynamic simulations revealed that biflavonoids decrease the content of ß-sheet in Aß fibril in a structure-dependent manner. Hydrogen bond analysis further supported that the substitution of hydroxyl groups capable of hydrogen bond formation at two positions on the biflavonoid scaffold leads to significantly disaggregation of Aß fibrils. Taken together, our data indicate that biflavonoids promote disaggregation of Aß fibrils due to their ability to disrupt the fibril structure, suggesting biflavonoids as a lead class of compounds to develop a therapeutic agent for Alzheimer's disease.

Are infectious reproductive pathogens of large ruminants a threat to improving food security? An investigation from Cambodia.

In Cambodia, information on common pathogens causing reproductive losses in cattle and buffalo are lacking, despite there being a need to address livestock health to enhance food security. We analysed stored buffalo (n = 29) and cattle (n = 471) serum samples collected in 2016 using commercially available enzyme-linked immunosorbent assay kits. Antibodies to Neospora caninum, bovine viral diarrhoea virus (BVDV), Leptospira interrogans serovar Hardjo and Brucella abortus were detected in buffalo samples at 79.3% (95% CI 64.6-94.0), 3.4% (95% CI 0-10.0), 0% and 0%, respectively, and in cattle at 4.2% (95% CI 2.4-6.0), 6.4% (95% CI 4.2-8.6), 8.1% (95% CI 5.6-10.6) and 0%, respectively. The high N. caninum seroprevalence in buffalo was associated with increasing age, with buffalo having a 13.1% chance of being seropositive at birth, increasing to 99.4% by age 7 (p = 0.045). This suggests a predominance of horizontal transmission, possibly from exposure to faeces from free-roaming village dogs. Cattle L. interrogans serovar Hardjo seroprevalence was highest in Tbong Khmum province (20.4%) compared to other provinces (p < 0.001), and may be compromising bovine fertility and creating a zoonotic risk for smallholders who may contract leptospirosis from farm work. These high infection rates prompt further research to determine: to what extent these pathogens are linked to the low reproductive efficiency observed in large ruminants in Cambodia, the risk factors to pathogen exposure and appropriate strategies to reduce these risks. Low BVDV and B. abortus exposure is an important observation. Increasing large ruminant livestock trade into the country will require improved biosecurity and disease surveillance to prevent their emergence. An enhanced understanding of the status of infectious reproductive livestock pathogens in Cambodia can assist development projects to make evidence-based strategies to enhance cattle and buffalo health and improve food security.

A history of FMD research and control programmes in Southeast Asia: lessons from the past informing the future.

Foot and mouth disease (FMD) is a major animal health problem within Southeast Asia (SEA). Although Indonesia and more recently the Philippines have achieved freedom from FMD, the disease remains endemic on continental SEA. Control of FMD within SEA would increase access to markets in more developed economies and reduce lost productivity in smallholder and emerging commercial farmer settings. However, despite many years of vaccination by individual countries, numerous factors have prevented the successful control of FMD within the region, including unregulated 'informal' transboundary movement of livestock and their products, difficulties implementing vaccination programmes, emergence of new virus topotypes and lineages, low-level technical capacity and biosecurity at national levels, limited farmer knowledge on FMD disease recognition, failure of timely outbreak reporting and response, and limitations in national and international FMD control programmes. This paper examines the published research of FMD in the SEA region, reviewing the history, virology, epidemiology and control programmes and identifies future opportunities for FMD research aimed at the eventual eradication of FMD from the region.

Innovative pain management solutions in animals may provide improved wound pain reduction during debridement in humans: An opinion informed by veterinary literature.

Painful animal husbandry procedures are routinely performed in a range of livestock species without analgesia. Recently, innovative strategies have been developed to address wound pain in these animals. In particular, a farmer-applied "spray and stay" approach that is administered directly to open wounds was developed (Tri-Solfen® Medical Ethics Pty Ltd., Melbourne, Victoria, Australia). This strategy anaesthetises the wounds immediately upon their formation, with long-lasting effect. This development, described as a "pain management revolution," has become firmly established in the Australian livestock industries and has global potential. The positive outcomes of this approach provide insights and highlight potential benefits that may be accrued from its use in human wound care, providing rapid-onset wound analgesia and/or anaesthetising wounds prior to cleansing and debridement procedures. If these benefits are realised from a clinician and patient perspective for wound debridement as an initial indication, it could provide new horizons in pain management for a spectrum of wound-related procedures. Evidence from use in animal husbandry does support the concept that multimodal anaesthesia holds great potential in the field of wound management across many procedures.

Livestock and livelihoods of smallholder cattle-owning households in Cambodia: the contribution of on-farm and off-farm activities to income and food security.

The majority of smallholder farming households in Cambodia are rurally based and rely on agriculture to support their livelihoods. However, in recent years, growth in the agriculture sector has stagnated with farmers facing several challenges including declining prices for traditional crops and irregular rainfall patterns. This has led to a need for farmers to diversify income sources with livestock promoted as a more viable livelihood activity, particularly the raising of cattle and poultry. However, uncertain profitability of livestock activities is a common perception by smallholders, especially where animals have not been traditionally viewed as a primary income source. To address this, information is required which compares the income and expenses associated with livestock raising to other on-farm activities and off-farm sources. This study reports on a survey of livelihood survey of 17 male and 21 female representatives of 20 households owning cattle in Cambodia, comparing the associated income and expenses of the various livelihood activities in the 12-month period from January to December 2016. Combined total household income from both on-farm and off-farm sources ranged from USD875 to 17730 with an average of USD6779, representing 51% and 49% from on-farm and off-farm activities, respectively. Cattle raising was the most common source of on-farm income (85%), contributing USD1064 and representing 22% of total household income. General household expenses, such as food and transportation (including expenses associated with off-farm employment), represented the majority of total household expenses (79%). Gross profit calculations indicated higher values for pig raising (USD1841.79), cattle (USD950.80) and non-rice crops (USD884) whilst the highest gross margin value was recorded for cattle (89.33%) followed by vegetables (85.27%) and non-rice crops (83.08%). Whilst wet season and dry season rice returned a negative gross profit value of USD197.27 and USD90.60 on average per household, respectively, both were ranked as most important for household consumption, followed by poultry, providing the main source of energy (rice) and protein (chicken meat) and sustaining household food requirements. The study concludes that although smallholder cattle-owning households in Cambodia undertake a diverse range of on-farm activities, as cattle raising provides a superior income source due to higher returns and lower variable costs, it could be promoted as a preferred livelihood activity by agencies conducting rural extension activities. Although consideration of available labour and monetary funds to invest in cattle raising is required, it was observed that income from off-farm sources may prove beneficial in providing the additional monetary funds to support cattle-raising activities and assist in providing generally poor smallholder households with enhanced economic resilience.

Effect of topical vapocoolant spray on perioperative pain response of unweaned calves to ear tagging and ear notching.

OBJECTIVE: To evaluate the efficacy of a vapocoolant spray to provide local anaesthesia for calves during ear tagging and ear notching. STUDY DESIGN: Randomized study. ANIMALS: Twenty Black Angus calves aged 4-16 weeks old. METHODS: Temperature validation studies using thermocouples and a temperature data logger were conducted on dead and live tissue to determine optimal spray distance and duration to achieve tissue anaesthesia (<10 °C). A behavioural trial was conducted to assess efficacy for ear tagging and ear notching. Calves (n = 20) were randomly assigned to the vapocoolant spray (VS) or the water spray (WS) group. A 3 second spray was administered from 10 cm to both sides of the ear immediately prior to ear tagging and ear notching. A numerical rating scale (NRS) was used to score behavioural response to each procedure, with responses categorized from 0 (no response) to 3 (severe). RESULTS: Temperature and tissue validation studies indicated that a vapocoolant spray reduced dead and live tissue temperature to below nociceptive threshold levels (10 °C) for 10-16 seconds. Univariate analysis indicated that ear notching was more painful than ear tagging [odds ratio (OR) = 19.2, 95% confidence interval (CI): 5.34-68.99, p < 0.001]. When adjusted for the multivariate model, there was a significant effect of treatment, with WS calves showing higher pain response scores than VS calves (OR = 4.08, 95% CI: 1.34-12.42, p = 0.011). Ear notching resulted in greater pain response scores than ear tagging (OR = 23.19, 95% CI: 6.18-87.05, p < 0.001). CONCLUSIONS: and clinical relevance Vapocoolant spray induced local anaesthesia and significantly reduced the pain response to ear tagging and ear notching in calves. Ear notching is more painful than ear tagging. Cryoanaesthesia is an effective option for reducing the perioperative pain associated with these simple husbandry procedures.

Investigating baseline red meat slaughter operator capacity and directions for development in Lao PDR.

A study of operator knowledge, attitudes and practices (KAP) in red meat slaughter premise operations in northern Laos was conducted and compared with international best practice, to inform future industry development. The survey interviewed 68 (of 94 possible participants) employees from all ten commercial slaughter premises in six districts in three northern Laos provinces. This was supported by observations of slaughter premises, processes and the conduct of personnel. Descriptive analysis and linear regression modelling identified significant KAP predictor factors, and a gap analysis supported or rejected inferences from the generally low KAP scores for human and animal health, animal welfare, good manufacturing practices (GMP), work conditions and economics. The median proportion of correctly/desirably answered knowledge-related questions was 35.2% (interquartile range [IQR] = 22.2-51.9%) with 27.3% (IQR = 15.9-31.8%) for the attitude-related questions and 21.4% (IQR = 14.3-35.7%) for the practice-related questions. Two districts had significantly lower KAP scores than other districts, and staff had the lowest and meat inspectors had the highest scores. This study indicates that the current KAP for red meat processing falls short of international standards and that training programmes on disease risks and prevention are important in facilitating red meat industry development.

Biliatresone, a Reactive Natural Toxin from Dysphania glomulifera and D. littoralis: Discovery of the Toxic Moiety 1,2-Diaryl-2-Propenone.

We identified a reactive natural toxin, biliatresone, from Dysphania glomulifera and D. littoralis collected in Australia that produces extrahepatic biliary atresia in a zebrafish model. Three additional isoflavonoids, including the known isoflavone betavulgarin, were also isolated. Biliatresone is in the very rare 1,2-diaryl-2-propenone class of isoflavonoids. The α-methylene of the 1,2-diaryl-2-propenone of biliatresone spontaneously reacts via Michael addition in the formation of water and methanol adducts. The lethal dose of biliatresone in a zebrafish assay was 1 µg/mL, while the lethal dose of synthetic 1,2-diaryl-2-propen-1-one was 5 µg/mL, suggesting 1,2-diaryl-2-propenone as the toxic Michael acceptor.

Survey of smallholder beef cattle production systems in different agro-ecological zones of Cambodia.

A survey was conducted to better understand the contribution of farm productivity to rural household income and identify differences in production systems, feeding practices and development constraints to smallholder beef cattle producers in the four agro-ecological zones (AEZs) of Cambodia. A semi-structured questionnaire was used to interview 360 households in the four AEZs: I, the Great Lake Floodplain; II, the Mekong Floodplain; III, the Coastal and IV, the Plateau/Mountainous. In addition, samples of common nutritional resources used for cattle feed were collected for nutrient composition analysis, plus cattle were scored for body condition. Rice farming and cattle production were the most common sources of income in all AEZs. The average cattle herd size was 3.7 (SD = 2.4), but the majority of households raised 1-3 animals. The most common cattle management system was grazing with supplementation, mainly with rice straw and 'cut-and-carry' natural grasses fed during the wet season in all AEZs. The body condition score of all cattle types was 3.2 (SD = 0.8), except for cows in lactation that were 1.8. Major constraints to cattle production in AEZs I, II and III were lack of quality feed resources, capital for cattle production and concerns on breed quality, whereas in AEZ IV, diseases were identified as the main constraint. This survey confirms the importance of cattle to smallholders in the four AEZs. Interventions including farmer education to improve husbandry skills, increase the utilisation of forages and crop residues and address disease issues are necessary to enhance cattle production and rural livelihoods in Cambodia.

Improving smallholder food security through investigations of carcass composition and beef marketing of buffalo and cattle in northern Lao PDR.

This study determined the carcass composition of Lao indigenous buffalo (Bubalus bubalis) and cattle (Bos indicus), then examined trends in bovine meat marketing following review of records of beef production and prices in the two major cities of Luang Prabang (LPB) and Xieng Khoung (XK) provinces in northern Laos. Samples from 41 buffalo and 81 cattle (n = 122) were collected from animals slaughtered in May-June 2014, with live weights, carcass weights and other carcass-related variables collected. The animals were classified into four age cohort groups (<2, 2-<4, 4-6 and >6 years) with quantitative and dichotomous qualitative traits determined. There were significant differences in buffalo and cattle predicted mean carcass weights between age classification categories (p = 0.003 and 0.001) but not in dressing percentages (p = 0.1 and 0.1). The carcass weight of buffalo was 104 (±23.1)-176 (±12.0) kg compared to 65 (±8.7)-84 (±6.5) kg of cattle, with dressing percentages of 37-40 and 39-42 %, respectively. Despite an average bovine meat price increase of 42-48 % between 2011 and 2013, there was a reduction in the numbers of large ruminants slaughtered in the surveyed cities of LPB (11 %) and XK (7 %), with bovine meat availability per person of 5.2-6.6 kg (LPB) and 3.0-3.8 kg (XK). Improving the sustainability of the bovine meat supply in Laos requires a systems approach involving improvements to animal health and production, livestock marketing, plus the critical development of improved slaughterhouse facilities enabling a meat-processing sector to emerge. This development pathway is of particular importance for building the capacity of Laos to reduce food insecurity and alleviate the poverty of its largely rural smallholder community.

Promoting transboundary animal disease risk management via a multiple health and husbandry intervention strategies in upland Lao PDR.

Surveys of smallholder farmer knowledge, attitude, practice (KAP) and income were conducted in 2011 and 2012 in five northern provinces of Lao People's Democratic Republic (Lao PDR; n = 200 farmers). Participating villages were classified as either from a livelihood development project (LV; n = 80 farmers) or a livestock research project that contained both 'high-intervention' (HI; n = 60 farmers) or 'low-intervention' (LI; n = 60 farmers) villages. Farmer training plus a suite of health and productivity interventions were implemented in HI sites; only vaccination was implemented in LI sites, whilst various interventions and limited vaccination were introduced to LV sites. Farmer interviews were conducted with survey questions on socioeconomic variables and KAP of large ruminant health and disease risk management, enabling determination of quantitative and dichotomous qualitative traits and comparison of results from HI, LI and LV villages. The average farmer income from sales of large ruminants in HI was USD 621, in LI was USD 547 and in LV was USD 225 (p < 0.001). The predicted mean of total knowledge scores (/42) in the 2012 survey in HI was 28, in LI was 22 and in LV was 17 (p < 0.001). We conclude that improved KAP of large ruminant health and production can be achieved by intensive training, although with some farmers yet to apply their knowledge on husbandry and biosecurity practices, continued learning support and closed linkage of research and development projects to improve extension capacity is recommended. This multiple participatory approach promoting biosecurity in addition to vaccination may provide a more sustainable pathway for the advancement of Lao PDR on the Progressive Control Pathway for foot and mouth disease control.

Use of a local anaesthetic and antiseptic wound formulation for the treatment of lambs naturally infected with Orf virus.

Contagious ecthyma (CE) is a worldwide highly contagious zoonotic viral skin disease of sheep and goats. Treatment for Orf virus (ORFV) infection usually involves topical and oral antibiotics. An anaesthetic and antiseptic topical gel (Multisolfen® or Tri-Solfen®; MS®, Medical Ethics, Australia) has been documented as an efficacious therapy for lesions from mucosal and epithelial viral infections in ruminants. The present study tested a new treatment protocol of MS® for CE therapy on-farm in 150 lambs naturally infected with ORFV. Lambs were divided into three cohorts of 50 lambs each (C, D and E). Cohort C was treated with MS® 3 times with an interval of 3 days between treatments, cohort D was treated daily with hypochlorous acid, whilst cohort E served as untreated controls. The lambs were examined clinically every two days, weight measured weekly, with whole blood and sterile swabs from ORFV lesions collected for haematological analysis and specific ORFV PCR. Cohort C presented fewer lambs displaying ORFV-associated lesions than other cohorts at different times of the experiment. Further, lesions treated with MS® were milder compared with other cohorts. However, following cessation of therapy, most of the lambs again developed ORFV-associated lesions. No differences between cohorts were observed in weight, haematological and PCR results. These findings suggest that topical treatment with MS® is effective for CE in field conditions, especially in the first stages of the clinical course, although treatment with MS® may need to be extended a minimum of 4 weeks.

Prevalence and clinical impact of Toxocara vitulorum in cattle and buffalo calves in northern Lao PDR.

This study was completed to determine the prevalence and distribution of Toxocara vitulorum infection in cattle and buffalo calves and investigate its clinical impact in northern Lao PDR (Peoples Democratic Republic). The results aim to assist decisions on disease control measures that can contribute to increasing cattle and buffalo productivity within smallholder farming systems in tropical areas. A prevalence survey for T. vitulorum in buffalo and cattle calves aged <3 months was conducted between September 2009 and June 2010 in five provinces of northern Lao PDR using a two-stage sampling technique to select 69 villages and 899 calves, with faecal samples collected and examined for T. vitulorum eggs at a local laboratory. At the time of sampling, data on calf morbidity and anthelmintic treatment was also collected. Factors potentially associated with infection and severity of infection were analyzed at univariable and multivariable levels, using T. vitulorum status (positive/negative) and on the positive calves only, faecal egg count levels as outcome variables. The estimated prevalence of T. vitulorum in northern Lao was 22.6 % (95 % confidence interval [CI], 0.17-0.28), and 76.8 % of villages had at least one positive calf. Province was the only significant (p < 0.05) variable investigated associated with calf infection status. Species (buffalo) was the only variable significantly (p < 0.05) associated with higher egg per gram of faeces levels among infected calves. Prevalence in calves aged 1-21 days, the reported prepatent period, was 17.5 % (CI 0.11-0.24). Treatment levels were very low (8.2 %) and if treatment occurred it was mostly unsuccessful. The high and wide spread infection of T. vitulorum in cattle and buffalo calves identified in this survey is likely to result in suboptimal cattle and buffalo productivity. Improved management of T. vitulorum infection in cattle and buffalo calves in northern Lao PDR is indicated to reduce potential negative production impacts and enable more efficient development of large ruminant livestock industry as a pathway from rural poverty for smallholder farmers in northern Lao PDR. In addition to quantifying this disease problem in calves, the conduct of this applied participatory research study provided an important opportunity to improve animal health services by increasing the parasite, large ruminant handling and research knowledge and capacity of government animal health staff and farmers.

Understanding ATP binding to DosS catalytic domain with a short ATP-lid.

DosS is a heme-sensor histidine kinase that responds to redox-active stimuli in mycobacterial environments by triggering dormancy transformation. Sequence comparison of the catalytic ATP-binding (CA) domain of DosS to other well-studied histidine kinases suggests that it possesses a rather short ATP-lid. This feature has been thought to inhibit DosS kinase activity by blocking ATP binding in the absence of interdomain interactions with the dimerization and histidine phospho-transfer (DHp) domain of full-length DosS. Here, we use a combination of computational modeling, structural biology, and biophysical studies to re-examine ATP-binding modalities in DosS's CA domain. We show that the closed lid conformation observed in protein crystal structures of DosS CA is caused by the presence of a zinc cation in the ATP binding pocket that coordinates with a glutamate residue on the ATP-lid. Furthermore, circular dichroism (CD) studies and comparisons of DosS CA crystal structure with its AlphaFold model and homologous DesK reveal that a key N-box alpha-helix turn of the ATP pocket manifests as a random coil in the zinc-coordinated protein crystal structure. We note that this closed lid conformation and the random-coil transformation of an N-box alpha-helix turn are artifacts arising from the millimolar zinc concentration used in DosS CA crystallization conditions. In contrast, in the absence of zinc, we find that the short ATP-lid of DosS CA has significant conformational flexibility and can bind ATP (Kd = 53 ± 13 µM). We conclude that DosS CA is almost always bound to ATP under physiological conditions (1-5 mM ATP, sub-nanomolar free zinc) in the bacterial environment. Our findings elucidate the conformational adaptability of the short ATP-lid, its relevance to ATP binding in DosS CA and provide insights that extends to 2988 homologous bacterial proteins containing such ATP-lids.

Gas tunnel engineering of prolyl hydroxylase reprograms hypoxia signaling in cells.

Molecular engineering of biocatalysts has revolutionized complex synthetic chemistry and sustainable catalysis. Here, we show that it is also possible to use engineered biocatalysts to reprogram signal transduction in human cells. More specifically, we manipulate cellular hypoxia (low O2) signaling by engineering the gas-delivery tunnel of prolyl hydroxylase 2 (PHD2), an iron-dependent enzymatic O2 sensor. Using computational modeling and rational protein design techniques, we resolve PHD2's gas tunnel and critical residues therein that limit the flow of O2 to PHD2's catalytic core. Systematic modification of these residues open the constriction topology of PHD2's gas tunnel with the most effectively designed mutant displaying 11-fold enhanced hydroxylation efficiency. Furthermore, transfection of plasmids that express these engineered PHD2 mutants in HEK-293T cells reveal significant reduction in the levels of hypoxia inducible factor (HIF-1α) even under hypoxic conditions. Our studies reveal that activated PHD2 mutants can reprogram downstream HIF pathways in cells to simulate physiological O2-like conditions despite extreme hypoxia and underscores the potential of engineered biocatalysts in controlling cellular function.

Use of a Local Anaesthetic/Antiseptic Formulation for the Treatment of Lambs Experimentally Infected with Orf Virus.

Contagious ecthyma is a highly transmissible eruptive viral disease of the skin and mucosa of sheep and goats distributed worldwide. The treatment of orf lesions is usually based on the use of antiseptics and antibiotics for the management of presumptive secondary infections, increasing risks of antimicrobial resistance. The wound dressing formulation Tri-Solfen® (TS) containing two local anaesthetics (lignocaine and bupivacaine), adrenaline and an antiseptic (cetrimide) in a gel formulation has been demonstrated to reduce suffering and enhance recovery in cattle and buffalo with oral and skin lesions due to foot-and-mouth disease virus infection and reduced the orf viral load in lambs. In the present study, experimental infection with the orf virus was conducted in 50 newborn lambs and 25 animals were treated after the presence of the first lesions with TS and repeated three days later. Daily clinical examination, haematological, serological, biomolecular and post-mortem analyses were conducted during 34 days after treatment. Results indicated that treatment had no effect on weight gain and clinical progression of the lesions. It was determined that seroconversion after experimental infection occurs 34 days after infection and suggested that the deep basal epithelial location of the orf lesions may have prevented the therapy from having altered the clinical course.