Design and development of an IoT-based trolley for weighing the patient in lying condition.

Introduction: An immobile patient cannot be weighed on a stand-on weighing machine, i.e., a bathroom scale. They have to get weighed while lying, which is not easy. The main objective of this research is to design a medical apparatus that measures the patient's weight in a lying condition. To achieve this the apparatus is designed as a stretcher to carry the patient in and around the hospital. Methods: The stretcher has four load cells to measure the patient's weight; it can bear a weight of 500 kg and has a self-weight of 20 kg. A Microcontroller unit (MCU) is embedded into the apparatus to weigh the patient lying on it. The stretcher comprises the top frame, middle frame, and base frame. The top frame can be detached and mounted back to the middle frame; this will help the medical personnel shift the patients from a medical bed. The middle frame is a plate structure where the four load cells are mounted at the corners of the lower plate. The upper plate functions as a pressure plate on the load cell. The base plate has four heavy-duty wheels that can bear the load. The middle frame and base frame, together, form a single structure, giving mobility to the structure. A control panel is employed with reset, tare, and on-off buttons to control the embedded platform. The LCD panel on the side of the apparatus shows the weight when the patient is placed on top of the apparatus. Results and discussion: A prototype trolley equipped with a wireless data logging system was tested on 10 healthy participants. The device accurately measured weight within ±50 g across a scale range of 2-140 kg, with data captured every 30 s over a 5-min testing period. Wireless communication was successfully demonstrated over a 100-m range. The important add-on feature of this work is the apparatus is connected to the internet, transforming it into an IoT-based medical device.

Functional implications of unusual NOS and SONOS covalent linkages found in proteins.

The tertiary and quaternary structures of many proteins are stabilized by strong covalent forces, of which disulfide bonds are the most well known. A new type of intramolecular and intermolecular covalent bond has been recently reported, consisting of the Lys and Cys side-chains linked by an oxygen atom (NOS). These post-translational modifications are widely distributed amongst proteins, and are formed under oxidative conditions. Similar linkages are observed during antibiotic biosynthesis, where hydroxylamine intermediates are tethered to the sulfur of enzyme active site Cys residues. These linkages open the way to understanding protein structure and function, give new insights into enzyme catalysis and natural product biosynthesis, and offer new strategies for drug design.

Advances in the structural basis for angiotensin-1 converting enzyme (ACE) inhibitors.

Human somatic angiotensin-converting enzyme (ACE) is a key zinc metallopeptidase that plays a pivotal role in the renin-angiotensin-aldosterone system (RAAS) by regulating blood pressure and electrolyte balance. Inhibition of ACE is a cornerstone in the management of hypertension, cardiovascular diseases, and renal disorders. Recent advances in structural biology techniques have provided invaluable insights into the molecular mechanisms underlying ACE inhibition, facilitating the design and development of more effective therapeutic agents. This review focuses on the latest advancements in elucidating the structural basis for ACE inhibition. High-resolution crystallographic studies of minimally glycosylated individual domains of ACE have revealed intricate molecular details of the ACE catalytic N- and C-domains, and their detailed interactions with clinically relevant and newly designed domain-specific inhibitors. In addition, the recently elucidated structure of the glycosylated form of full-length ACE by cryo-electron microscopy (cryo-EM) has shed light on the mechanism of ACE dimerization and revealed continuous conformational changes which occur prior to ligand binding. In addition to these experimental techniques, computational approaches have also played a pivotal role in elucidating the structural basis for ACE inhibition. Molecular dynamics simulations and computational docking studies have provided atomic details of inhibitor binding kinetics and energetics, facilitating the rational design of novel ACE inhibitors with improved potency and selectivity. Furthermore, computational analysis of the motions observed by cryo-EM allowed the identification of allosteric binding sites on ACE. This affords new opportunities for the development of next-generation allosteric inhibitors with enhanced pharmacological properties. Overall, the insights highlighted in this review could enable the rational design of novel ACE inhibitors with improved efficacy and safety profiles, ultimately leading to better therapeutic outcomes for patients with hypertension and cardiovascular diseases.

Effect of Home-Based Pulmonary Rehabilitation on Pulmonary Fibrosis.

BACKGROUND: Pulmonary fibrosis is a chronic, progressive lung condition that involves lung tissue scarring and thickening. The effects of home-based pulmonary rehabilitation (PR) in post-covid pulmonary fibrosis (PCPF) and other forms of fibrosis together have not been evaluated. This study aims to evaluate the effectiveness of home-based pulmonary rehabilitation on pulmonary function, functional capacity, and health-related quality of life in people with pulmonary fibrosis (post-COVID pulmonary fibrosis, pulmonary fibrosis secondary to pulmonary tuberculosis (TB), pulmonary fibrosis secondary to interstitial lung disease (ILD), pulmonary fibrosis secondary to bronchiectasis). METHODS: A single-group pretest-posttest experimental study was performed after recruiting 98 pulmonary fibrosis subjects from K.M.C hospitals. After being screened for the inclusion and exclusion criteria, 45 subjects were analyzed, and 6 subjects were lost to follow-up.  A home-based pulmonary rehabilitation program was carried out for 8 weeks (warm-up, stretching exercises, aerobic exercise, strength training for upper limb and lower limb, breathing exercises mainly involved; others: energy saving techniques, controlled coughing techniques, dyspnea relieving positions). The program was supervised via weekly phone calls. Pulmonary function (Pulmonary function test), exercise capacity (6-minute walk test), dyspnea (modified Borg scale), and health-related quality of life (SF-36) were evaluated before and after the intervention. During the enrollment and after the 6-minute walk test, saturation of peripheral oxygen (SPO2) level was also evaluated pre-intervention and after the 8-weeks program. RESULTS: Pulmonary function [FVC(L) t = -12.52, p<0.05; FEV1(L) t = -2.56, p<0.05; FEV1/FVC t = 7.98, p<0.05 and DLCO (ml/min/mmHg) t = -5.13, p<0.05], 6MWD [MD 88.66; p<0.05] and HRQOL measured by SF-36 scores (p<0.05) were improved significantly. Both the baseline SPO2 level before the 6MWT [MD 1.07, p<0.05] and the SPO2 level after the 6MWT [MD 1.16, p<0.05] showed a significant improvement. The rating of perceived exertion(dyspnea) [MD 1.30, p<0.05] was reduced significantly after the 8-week program. CONCLUSION: Our study shows that home-based pulmonary rehabilitation is an effective option for improving lung function and physical functional capacity by reducing dyspnea perception and improving the saturation of peripheral oxygen (SPO2) level, and enhancing the quality of life in people with pulmonary fibrosis.

Smartphone microscopic method for imaging and quantification of microplastics in drinking water.

Analysis of microplastics in drinking water is often challenging due to smaller particle size and low particle count. In this study, we used a low cost and an easy to assemble smartphone microscopic system for imaging and quantitating microplastic particles as small as 20 µm. The system consisted of a spherical sapphire ball lens of 4 mm diameter attached to a smartphone camera as a major imaging component. It also involved pre-concentration of the sample using ZnCl2 solution. The spike recovery and limit of detection of the method in filtered distilled and deionized water samples (n = 9) were 55.6% ± 9.7% and 34 particles/L, respectively. Imaging performance of the microscopic system was similar to a commercial bright field microscopic system. The method was further implemented to examine microplastic particles in commercial bottled and jar water samples (n = 20). The particles count in bottled and jar water samples ranged from 0-91 particles/L to 0-130 particles/L, respectively. In both sample types, particles of diverse shape and size were observed. The particles collected from water samples were further confirmed by FTIR spectra (n = 36), which found 97% of the particles tested were made of plastic material. These findings suggested that the smartphone microscopic system can be implemented as a low-cost alternative for preliminary screening of microplastic in drinking water samples. RESEARCH HIGHLIGHTS: Ball lens based smartphone microscopic method was used for microplastic analysis. Particles of diverse shape and size were found in bottle and jar water samples.

Clinical usefulness of nebulized dexmedetomidine for conscious sedation in daycare flexible bronchoscopy in Southern India.

BACKGROUND: Sedative agents used in bronchoscopy require trained personnel to administer and monitor the patient. This increases the procedure cost, duration, and inpatient stay. Inhalational administration of sedative agents can be a practical solution to the issue. Dexmedetomidine in the inhalational form could give results similar to the intravenous form without significant adverse events. MATERIALS AND METHODS: The study is prospective, randomized, and double-blinded study. Patients needing bronchoscopy were randomized to receive the nebulized form of either dexmedetomidine or saline (0.9%) before bronchoscopy. The study parameters are assessed and recorded before, during, and after bronchoscopy. Data collected are analyzed using the SPSS software. DISCUSSION: The side effects limit using commonly administered sedation agents in bronchoscopy, such as midazolam, fentanyl, and dexmedetomidine. The nebulized dexmedetomidine is safe with proven efficacy when compared to the placebo. Proceduralist-administered conscious sedation reduces the overall cost and shortens inpatient stays. Attenuation of hemodynamic parameters by dexmedetomidine could be an advantage for the physician in reducing an untoward cardiac event. CONCLUSION: Dexmedetomidine in the nebulized form improves the comfort of patients during the procedure. It blunts the pressure response during bronchoscopy and could be a safer and cost-effective agent in its nebulized form for conscious sedation in bronchoscopy. The study is approved by the institutional ethics committee (IEC KMC MLR 10-2021-310).

α-Methylacyl-CoA Racemase from Mycobacterium tuberculosis-Detailed Kinetic and Structural Characterization of the Active Site.

α-Methylacyl-CoA racemase in M. tuberculosis (MCR) has an essential role in fatty acid metabolism and cholesterol utilization, contributing to the bacterium's survival and persistence. Understanding the enzymatic activity and structural features of MCR provides insights into its physiological and pathological significance and potential as a therapeutic target. Here, we report high-resolution crystal structures for wild-type MCR in a new crystal form (at 1.65 Å resolution) and for three active-site mutants, H126A, D156A and E241A, at 2.45, 1.64 and 1.85 Å resolutions, respectively. Our analysis of the new wild-type structure revealed a similar dimeric arrangement of MCR molecules to that previously reported and details of the catalytic site. The determination of the structures of these H126A, D156A and E241A mutants, along with their detailed kinetic analysis, has now allowed for a rigorous assessment of their catalytic properties. No significant change outside the enzymatic active site was observed in the three mutants, establishing that the diminution of catalytic activity is mainly attributable to disruption of the catalytic apparatus involving key hydrogen bonding and water-mediated interactions. The wild-type structure, together with detailed mutational and biochemical data, provide a basis for understanding the catalytic properties of this enzyme, which is important for the design of future anti-tuberculosis drug molecules.

Structural insights into the inhibitory mechanism of angiotensin-I-converting enzyme by the lactotripeptides IPP and VPP.

Human somatic angiotensin-1-converting enzyme (sACE) is composed of a catalytic N-(nACE) and C-domain (cACE) of similar size with different substrate specificities. It is involved in the regulation of blood pressure by converting angiotensin I to the vasoconstrictor angiotensin II and has been a major focus in the development of therapeutics for hypertension. Bioactive peptides from various sources, including milk, have been identified as natural ACE inhibitors. We report the structural basis for the role of two lacototripeptides, Val-Pro-Pro and Ile-Pro-Pro, in domain-specific inhibition of ACE using X-ray crystallography and kinetic analysis. The lactotripeptides have preference for nACE due to altered polar interactions distal to the catalytic zinc ion. Elucidating the mechanism of binding and domain selectivity of these peptides also provides important insights into the functional roles of ACE.

Recombinant protein production for structural and kinetic studies: A case study using M. tuberculosis α-methylacyl-CoA racemase (MCR).

Modern drug discovery is a target-driven approach in which a particular protein such as an enzyme is implicated in the disease process. Commonly, small-molecule drugs are identified using screening, rational design, and structural biology approaches. Drug screening, testing and optimization is typically conducted in vitro, and copious amounts of protein are required. The advent of recombinant DNA technologies has resulted in a rise in proteins purified by affinity techniques, typically by incorporating an "affinity tag" at the N- or C-terminus. Use of these tagged proteins and affinity techniques comes with a host of issues. This chapter describes the production of an untagged enzyme, α-methylacyl-CoA racemase (MCR) from Mycobacterium tuberculosis, using a recombinant E. coli system. Purification of the enzyme on a 100 mg scale using tandem anion-exchange chromatographies (DEAE-sepharose and RESOURCE-Q columns), and size-exclusion chromatographies is described. A modified protocol allowing the purification of cationic proteins is also described, based on tandem cation-exchange chromatographies (using CM-sepharose and RESOURCE-S columns) and size-exclusion chromatographies. The resulting MCR protein is suitable for biochemical and structural biology applications. The described protocols have wide applicability to the purification of other recombinant proteins and enzymes without using affinity chromatography.

Engineering Plastic Eating Enzymes Using Structural Biology.

Plastic pollution has emerged as a significant environmental concern in recent years and has prompted the exploration of innovative biotechnological solutions to mitigate plastic's negative impact. The discovery of enzymes capable of degrading specific types of plastics holds promise as a potential solution. However, challenges with efficiency, industrial scalability, and the diverse range of the plastic waste in question, have hindered their widespread application. Structural biology provides valuable insights into the intricate interactions between enzymes and plastic materials at an atomic level, and a deeper understanding of their underlying mechanisms is essential to harness their potential to address the mounting plastic waste crisis. This review article examines the current biochemical and biophysical methods that may facilitate the development of enzymes capable of degrading polyethylene terephthalate (PET), one of the most extensively used plastics. It also discusses the challenges that must be addressed before substantial advancements can be achieved in using these enzymes as a solution to the plastic pollution problem.

Crystal Structure of the Catalytic Domain of a Botulinum Neurotoxin Homologue from Enterococcus faecium: Potential Insights into Substrate Recognition.

Clostridium botulinum neurotoxins (BoNTs) are the most potent toxins known, causing the deadly disease botulism. They function through Zn2+-dependent endopeptidase cleavage of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, preventing vesicular fusion and subsequent neurotransmitter release from motor neurons. Several serotypes of BoNTs produced by Clostridium botulinum (BoNT/A-/G and/X) have been well-characterised over the years. However, a BoNT-like gene (homologue of BoNT) was recently identified in the non-clostridial species, Enterococcus faecium, which is the leading cause of hospital-acquired multi-drug resistant infections. Here, we report the crystal structure of the catalytic domain of a BoNT homologue from Enterococcus faecium (LC/En) at 2.0 Å resolution. Detailed structural analysis in comparison with the full-length BoNT/En AlphaFold2-predicted structure, LC/A (from BoNT/A), and LC/F (from BoNT/F) revealed putative subsites and exosites (including loops 1-5) involved in recognition of LC/En substrates. LC/En also appears to possess a conserved autoproteolytic cleavage site whose function is yet to be established.

Molecular basis of selective amyloid-ß degrading enzymes in Alzheimer's disease.

The accumulation of the small 42-residue long peptide amyloid-ß (Aß) has been proposed as a major trigger for the development of Alzheimer's disease (AD). Within the brain, the concentration of Aß peptide is tightly controlled through production and clearance mechanisms. Substantial experimental evidence now shows that reduced levels of Aß clearance are present in individuals living with AD. This accumulation of Aß can lead to the formation of large aggregated amyloid plaques-one of two detectable hallmarks of the disease. Aß-degrading enzymes (ADEs) are major players in the clearance of Aß. Stimulating ADE activity or expression, in order to compensate for the decreased clearance in the AD phenotype, provides a promising therapeutic target. It has been reported in mice that upregulation of ADEs can reduce the levels of Aß peptide and amyloid plaques-in some cases, this led to improved cognitive function. Among several known ADEs, neprilysin (NEP), endothelin-converting enzyme-1 (ECE-1), insulin degrading enzyme (IDE) and angiotensin-1 converting enzyme (ACE) from the zinc metalloprotease family have been identified as important. These ADEs have the capacity to digest soluble Aß which, in turn, cannot form the toxic oligomeric species. While they are known for their amyloid degradation, they exhibit complexity through promiscuous nature and a broad range of substrates that they can degrade. This review highlights current structural and functional understanding of these key ADEs, giving some insight into the molecular interactions that leads to the hydrolysis of peptide substrates, the crucial tasks performed by them and the potential for therapeutic use in the future.

A Molecular Analysis of the Aminopeptidase P-Related Domain of PID-5 from Caenorhabditis elegans.

A novel protein, PID-5, has been shown to be a requirement for germline immortality and has recently been implicated in RNA-induced epigenetic silencing in the Caenorhabditis elegans embryo. Importantly, it has been shown to contain both an eTudor and aminopeptidase P-related domain. However, the silencing mechanism has not yet been fully characterised. In this study, bioinformatic tools were used to compare pre-existing aminopeptidase P molecular structures to the AlphaFold2-predicted aminopeptidase P-related domain of PID-5 (PID-5 APP-RD). Structural homology, metal composition, inhibitor-bonding interactions, and the potential for dimerisation were critically assessed through computational techniques, including structural superimposition and protein-ligand docking. Results from this research suggest that the metallopeptidase-like domain shares high structural homology with known aminopeptidase P enzymes and possesses the canonical 'pita-bread fold'. However, the absence of conserved metal-coordinating residues indicates that only a single Zn2+ may be bound at the active site. The PID-5 APP-RD may form transient interactions with a known aminopeptidase P inhibitor and may therefore recognise substrates in a comparable way to the known structures. However, loss of key catalytic residues suggests the domain will be inactive. Further evidence suggests that heterodimerisation with C. elegans aminopeptidase P is feasible and therefore PID-5 is predicted to regulate proteolytic cleavage in the silencing pathway. PID-5 may interact with PID-2 to bring aminopeptidase P activity to the Z-granule, where it could influence WAGO-4 activity to ensure the balanced production of 22G-RNA signals for transgenerational silencing. Targeted experiments into APPs implicated in malaria and cancer are required in order to build upon the biological and therapeutic significance of this research.

A silent march-Post covid fibrosis in asymptomatics - A cause for concern?

We report a case series of patients presenting with undiagnosed pulmonary fibrosis as a primary manifestation. On evaluation, after excluding other causes, the fibrosis was attributed to asymptomatic or mild COVID illness in the past. This case series serves to highlight the difficulties posed to clinicians while evaluating pulmonary fibrosis in the post-COVID era, more so in mild to asymptomatic COVID-19. The intriguing possibility of fibrosis setting even in mild to asymptomatic COVID is discussed.

17-year trends of body mass index, overweight, and obesity among adolescents from 2005 to 2021, including the COVID-19 pandemic: a Korean national representative study.

OBJECTIVE: There is a lack of pediatric studies that have analyzed trends in mean body mass index (BMI) and the prevalence of obesity and overweight over a period that includes the mid-stage of the COVID-19 pandemic. Thus, we aimed to investigate trends in BMI, overweight, and obesity among Korean adolescents from 2005 to 2021, including the COVID-19 pandemic. SUBJECTS AND METHODS: We used data from the Korea Youth Risk Behavior Web-based Survey (KYRBS), which is nationally representative of South Korea. The study included middle- and high-school students between the ages of 12 and 18. We examined trends in mean BMI and prevalence of obesity and/or overweight during the COVID-19 pandemic and compared these to those of pre-pandemic trends in each subgroup by gender, grade, and residential region. RESULTS: Data from 1,111,300 adolescents (mean age: 15.04 years) were analyzed. The estimated weighted mean BMI was 20.48 kg/m2 (95% CI, 20.46-20.51) between 2005 and 2007, and this was 21.61 kg/m2 (95% CI, 21.54-21.68) in 2021. The prevalence of overweight and obesity was 13.1% (95% CI, 12.9-13.3%) between 2005 and 2007 and 23.4% (95% CI, 22.8-24.0%) in 2021. The mean BMI and prevalence of obesity and overweight have gradually increased over the past 17 years; however, the extent of change in mean BMI and in the prevalence of obesity and overweight during the pandemic was distinctly less than before. The 17-year trends in the mean BMI, obesity, and overweight exhibited a considerable rise from 2005 to 2021; however, the slope during the COVID-19 pandemic (2020-2021) was significantly less prominent than in the pre-pandemic (2005-2019). CONCLUSIONS: These findings enable us to comprehend long-term trends in the mean BMI of Korean adolescents and further emphasize the need for practical prevention measures against youth obesity and overweight.

A Comprehensive Structural Analysis of Clostridium botulinum Neurotoxin A Cell-Binding Domain from Different Subtypes.

Botulinum neurotoxins (BoNTs) cause flaccid neuromuscular paralysis by cleaving one of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex proteins. BoNTs display high affinity and specificity for neuromuscular junctions, making them one of the most potent neurotoxins known to date. There are seven serologically distinct BoNTs (serotypes BoNT/A to BoNT/G) which can be further divided into subtypes (e.g., BoNT/A1, BoNT/A2…) based on small changes in their amino acid sequence. Of these, BoNT/A1 and BoNT/B1 have been utilised to treat various diseases associated with spasticity and hypersecretion. There are potentially many more BoNT variants with differing toxicological profiles that may display other therapeutic benefits. This review is focused on the structural analysis of the cell-binding domain from BoNT/A1 to BoNT/A6 subtypes (HC/A1 to HC/A6), including features such as a ganglioside binding site (GBS), a dynamic loop, a synaptic vesicle glycoprotein 2 (SV2) binding site, a possible Lys-Cys/Cys-Cys bridge, and a hinge motion between the HCN and HCC subdomains. Characterising structural features across subtypes provides a better understanding of how the cell-binding domain functions and may aid the development of novel therapeutics.

Crystal Structures of the Clostridium botulinum Neurotoxin A6 Cell Binding Domain Alone and in Complex with GD1a Reveal Significant Conformational Flexibility.

Clostridium botulinum neurotoxin A (BoNT/A) targets the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, by cleaving synaptosomal-associated protein of 25 kDa size (SNAP-25). Cleavage of SNAP-25 results in flaccid paralysis due to repression of synaptic transmission at the neuromuscular junction. This activity has been exploited to treat a range of diseases associated with hypersecretion of neurotransmitters, with formulations of BoNT/A commercially available as therapeutics. Generally, BoNT activity is facilitated by three essential domains within the molecule, the cell binding domain (HC), the translocation domain (HN), and the catalytic domain (LC). The HC, which consists of an N-terminal (HCN) and a C-terminal (HCC) subdomain, is responsible for BoNT's high target specificity where it forms a dual-receptor complex with synaptic vesicle protein 2 (SV2) and a ganglioside receptor on the surface of motor neurons. In this study, we have determined the crystal structure of botulinum neurotoxin A6 cell binding domain (HC/A6) in complex with GD1a and describe the interactions involved in ganglioside binding. We also present a new crystal form of wild type HC/A6 (crystal form II) where a large 'hinge motion' between the HCN and HCC subdomains is observed. These structures, along with a comparison to the previously determined wild type crystal structure of HC/A6 (crystal form I), reveals the degree of conformational flexibility exhibited by HC/A6.

Possible Consequences of Climate Change on Survival, Productivity and Reproductive Performance, and Welfare of Himalayan Yak (Bos grunniens).

Yak are adapted to the extreme cold, low oxygen, and high solar radiation of the Himalaya. Traditionally, they are kept at high altitude pastures during summer, moving lower in the winter. This system is highly susceptible to climate change, which has increased ambient temperatures, altered rainfall patterns and increased the occurrence of natural disasters. Changes in temperature and precipitation reduced the yield and productivity of alpine pastures, principally because the native plant species are being replaced by less useful shrubs and weeds. The impact of climate change on yak is likely to be mediated through heat stress, increased contact with other species, especially domestic cattle, and alterations in feed availability. Yak have a very low temperature humidity index (52 vs. 72 for cattle) and a narrow thermoneutral range (5-13 °C), so climate change has potentially exposed yak to heat stress in summer and winter. Heat stress is likely to affect both reproductive performance and milk production, but we lack the data to quantify such effects. Increased contact with other species, especially domestic cattle, is likely to increase disease risk. This is likely to be exacerbated by other climate-change-associated factors, such as increases in vector-borne disease, because of increases in vector ranges, and overcrowding associated with reduced pasture availability. However, lack of baseline yak disease data means it is difficult to quantify these changes in disease risk and the few papers claiming to have identified such increases do not provide robust evidence of increased diseases. The reduction in feed availability in traditional pastures may be thought to be the most obvious impact of climate change on yak; however, it is clear that such a reduction is not solely due to climate change, with socio-economic factors likely being more important. This review has highlighted the large potential negative impact of climate change on yak, and the lack of data quantifying that impact. More research on the impact of climate change in yak is needed. Attention also needs to be paid to developing mitigating strategies, which may include changes in the traditional system such as providing shelter and supplementary feed and, in marginal areas, increased use of yak-cattle hybrids.