Scbean: a python library for single-cell multi-omics data analysis.

SUMMARY: Single-cell multi-omics technologies provide a unique platform for characterizing cell states and reconstructing developmental process by simultaneously quantifying and integrating molecular signatures across various modalities, including genome, transcriptome, epigenome, and other omics layers. However, there is still an urgent unmet need for novel computational tools in this nascent field, which are critical for both effective and efficient interrogation of functionality across different omics modalities. Scbean represents a user-friendly Python library, designed to seamlessly incorporate a diverse array of models for the examination of single-cell data, encompassing both paired and unpaired multi-omics data. The library offers uniform and straightforward interfaces for tasks, such as dimensionality reduction, batch effect elimination, cell label transfer from well-annotated scRNA-seq data to scATAC-seq data, and the identification of spatially variable genes. Moreover, Scbean's models are engineered to harness the computational power of GPU acceleration through Tensorflow, rendering them capable of effortlessly handling datasets comprising millions of cells. AVAILABILITY AND IMPLEMENTATION: Scbean is released on the Python Package Index (PyPI) (https://pypi.org/project/scbean/) and GitHub (https://github.com/jhu99/scbean) under the MIT license. The documentation and example code can be found at https://scbean.readthedocs.io/en/latest/.

Highly Pathogenic Avian Influenza A(H5N1) Virus Clade 2.3.4.4b in Domestic Ducks, Indonesia, 2022.

Highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b viruses were isolated from domestic ducks in South Kalimantan, Indonesia, during April 2022. The viruses were genetically similar to those detected in East Asia during 2021-2022. Molecular surveillance of wild birds is needed to detect potential pandemic threats from avian influenza virus.

Design and Rationale for the Direct Oral Anticoagulant Apixaban in Left Ventricular Assist Devices (DOAC LVAD) Study.

Implantable left ventricular assist device (LVAD) therapy is used to improve quality of life, alleviate symptoms and extend survival rates in patients with advanced heart failure. Patients with LVADs require chronic anticoagulation to reduce the risk of thromboembolic complications, and they commonly experience bleeding events. Apixaban is a direct oral anticoagulant that has become first-line therapy for patients with nonvalvular atrial fibrillation and venous thromboembolism; however, its safety in patients with LVADs has not been well characterized. The evaluation of the hemocompatibility in the DOAC LVAD (Direct Oral Anti-Coagulant apixaban in Left Ventricular Assist Devices) trial is a phase 2, open-label trial of patients with LVADs who were randomized to either apixaban or warfarin therapy. Patients randomized to apixaban will be started on a dosage of 5 mg twice daily, whereas those randomized to warfarin will be managed at an International Normalized Ratio goal of 2.0-2.5. All patients will be treated with aspirin at 81 mg daily. We plan to randomize and follow as many as 40 patients for 24 weeks to evaluate the primary outcomes of freedom from death or hemocompatibility-related adverse events (stroke, device thrombosis, bleeding, aortic root thrombus, and arterial non-CNS thromboembolism). The DOAC LVAD trial will establish the feasibility of apixaban anticoagulant therapy in patients with LVADs. Clinicaltrials.gov: NCT04865978.

Training Pathways in Critical Care Cardiology: Competencies and Considerations for Cardiologists.

PURPOSE OF REVIEW: Critical care cardiology (CCC) is a rapidly developing field undergoing a renaissance of interest and growth to meet the well-documented population shift in the cardiac intensive care unit (CICU). With this has come the emergence of novel training paradigms that seek to combine specialties with meaningful overlap. RECENT FINDINGS: The benefit of having critical care expertise in the CICU has been clearly established; however, there is no formal or uniform CCC training pathway. Contemporary approaches seek to provide appropriate clinical and procedural experience while minimizing opportunity cost. The combination of additional cardiology subspecialties, specifically advanced heart failure or interventional cardiology, has been demonstrated. Educational tracks that integrate critical care training have generated interest but have not yet manifested. CCC training strives to meet the needs of an increasingly sick and diverse patient population while preparing trainees for fulfilling and meaningful careers. The hope is for ongoing development of novel training pathways to satisfy evolving needs.

New pattern of atypical advanced interatrial block.

INTRODUCTION: Interatrial block (IAB) is defined as prolonged P-wave duration (≥ 120 ms) due to delayed conduction in the Bachmann bundle. This is readily identifiable using surface electrocardiogram (ECG). Advanced IAB can be classified as typical and atypical. Atypical IAB can be further categorized by (i) duration or (ii) morphology. In this report, we have identified a new pattern of atypical IAB with triphasic morphology of the P-wave in the inferior leads. METHODS: Two clinical cases were evaluated including surface ECGs. P-wave durations and amplitudes were measured with digital calipers using ECG analysis software (MUSE, GE HealthCare). Comparisons were made using prior data to evaluate IAB and P-wave duration and morphology. RESULTS: A new pattern of atypical advanced IAB shows prolonged P-wave duration (P wave >160 ms) and triphasic morphology in all inferior leads with P +/+/- and P +/-/+, respectively. We speculate that triphasic P-waves in the inferior leads represent three moments of atrial depolarization; from right to left. CONCLUSION: This study describes a novel pattern of atypical advanced IAB. Further investigation regarding the increased risk of atrial fibrillation and stroke associated with this new pattern is warranted in the future.

Orange Pomace and Peel Extraction Processes towards Sustainable Utilization: A Short Review.

More than 58 million metric tonnes of oranges were produced in 2021, and the peels, which account for around one-fifth of the fruit weight, are often discarded as waste in the orange juice industry. Orange pomace and peels as wastes are used as a sustainable raw material to make valuable products for nutraceuticals. The orange peels and pomace contain pectin, phenolics, and limonene, which have been linked to various health benefits. Various green extraction methods, including supercritical carbon dioxide (ScCO2) extraction, subcritical water extraction (SWE), ultrasound-assisted extraction (UAE), and microwave-assisted extraction (MAE), are applied to valorize the orange peels and pomace. Therefore, this short review will give insight into the valorization of orange peels/pomace extraction using different extraction methods for health and wellness. This review extracts information from articles written in English and published from 2004 to 2022. The review also discusses orange production, bioactive compounds in orange peels/pomaces, green extractions, and potential uses in the food industry. Based on this review, the valorization of orange peels and pomaces can be carried out using green extraction methods with high quantities and qualities of extracts. Therefore, the extract can be used for health and wellness products.

Insight into Green Extraction for Roselle as a Source of Natural Red Pigments: A Review.

Roselle (Hibiscus sabdariffa L.) is a source of anthocyanins as red pigments that is extensively farmed in tropical and subtropical regions, including Indonesia, Malaysia, China, Thailand, Egypt, Mexico, and West India. The roselle plant contains a variety of nutrients, including anthocyanins, organic acids, pectin, etc. Due to the toxicity and combustibility of the solvents, traditional extraction methods for these compounds are restricted. Obtaining pure extracts is typically a lengthy procedure requiring many processes. Supercritical carbon dioxide (ScCO2) extraction as a green technology is rapidly improving and extending its application domains. The advantages of this method are zero waste production, quicker extraction times, and reduced solvent consumption. The ScCO2 extraction of natural pigments has great promise in food, pharmaceuticals, cosmetics, and textiles, among other uses. The ScCO2 technique for natural pigments may also be advantageous in a variety of other contexts. Due to their minimal environmental risk, the high-quality red pigments of roselle rich in anthocyanins extracted using ScCO2 extraction have a high sustainability potential. Therefore, the objective of this review is to increase knowledge related to the natural colorant of roselle as a substitute for chemically manufactured colorants using ScCO2 as a green method. This article covers ScCO2 extraction, particularly as it relates to the optimization of pigments that promote health. This article focuses on the high extraction efficiency of ScCO2 extraction. Natural colorants extracted via ScCO2 are regarded as safe compounds, especially for human consumption, such as novel functional food additives and textile and pharmaceutical colors.

Valorization of Peanut Skin as Agricultural Waste Using Various Extraction Methods: A Review.

Peanuts (Arachis hypogea) can be made into various products, from oil to butter to roasted snack peanuts and candies, all from the kernels. However, the skin is usually thrown away, used as cheap animal feed, or as one of the ingredients in plant fertilizer due to its little value on the market. For the past ten years, studies have been conducted to determine the full extent of the skin's bioactive substance repertoire and its powerful antioxidant potential. Alternatively, researchers reported that peanut skin could be used and be profitable in a less-intensive extraction technique. Therefore, this review explores the conventional and green extraction of peanut oil, peanut production, peanut physicochemical characteristics, antioxidant activity, and the prospects of valorization of peanut skin. The significance of the valorization of peanut skin is that it contains high antioxidant capacity, catechin, epicatechin resveratrol, and procyanidins, which are also advantageous. It could be exploited in sustainable extraction, notably in the pharmaceutical industries.

Considerations for Inotrope and Vasopressor Use in Critically Ill Patients With Pulmonary Arterial Hypertension.

ABSTRACT: Pulmonary arterial hypertension (PAH) is a rare and progressive cardiopulmonary disease, characterized by pulmonary vasculopathy. The disease can lead to increase pulmonary arterial pressures and eventual right ventricle failure due to elevated afterload. The prevalence of PAH in patients admitted to the intensive care unit (ICU) is unknown, and pulmonary hypertension (PH) in the ICU is more commonly the result of left heart disease or hypoxic lung injury (PH due to left heart disease and PH due to lung diseases and/or hypoxia, respectively), as opposed to PAH. Management of patients with PAH in the ICU is complex as it requires a careful balance to maintain perfusion while optimizing right-sided heart function. A comprehensive understanding of the underlying physiology and underlying hemodynamics is crucial for the management of this population. In this review, we summarized the evidence for use of vasopressors and inotropes in the management of PH and extrapolated the data to patients with PAH. We strongly believe that the understanding of the hemodynamic consequences of inotropes and vasopressors, especially from data in the PH population, can lead to better management of this complex patient population.

Heat Shock Alters the Proteomic Profile of Equine Mesenchymal Stem Cells.

The aim of this research was to determine the impact of heat stress on cell differentiation in an equine mesenchymal stem cell model (EMSC) through the application of heat stress to primary EMSCs as they progressed through the cell specialization process. A proteomic analysis was performed using mass spectrometry to compare relative protein abundances among the proteomes of three cell types: progenitor EMSCs and differentiated osteoblasts and adipocytes, maintained at 37 °C and 42 °C during the process of cell differentiation. A cell-type and temperature-specific response to heat stress was observed, and many of the specific differentially expressed proteins were involved in cell-signaling pathways such as Notch and Wnt signaling, which are known to regulate cellular development. Furthermore, cytoskeletal proteins profilin, DSTN, SPECC1, and DAAM2 showed increased protein levels in osteoblasts differentiated at 42 °C as compared with 37 °C, and these cells, while they appeared to accumulate calcium, did not organize into a whorl agglomerate as is typically seen at physiological temperatures. This altered proteome composition observed suggests that heat stress could have long-term impacts on cellular development. We propose that this in vitro stem cell culture model of cell differentiation is useful for investigating molecular mechanisms that impact cell development in response to stressors.

Bright as day and dark as night: light-dependant energy for lipid biosynthesis and production in microalgae.

Microalgae are photosynthetic organisms functioning as the green bio-factories for various pharmaceutical and biofuel products. To date, numerous attempts have been carried out to manipulate culture conditions to maximize the production of the desired metabolites. Because light is the energy source of microalgae for their growth and metabolites biosynthesis, it has been one of the most investigated variables emphasized on the deep understanding of how microalgae respond towards light changes as an external stimulus. This review discusses the effects of different light sources, light intensities, light wavelengths and length of photoperiod on various microalgae species, especially in terms of biomass and lipid productivity. Additionally, the relationship between photoregulation processes and lipid productivity of microalgae are also deliberated. The current available approaches of microalgae mass cultivation, including different types of open and closed systems are recapitulated with the intention to highlight the significant insights for the design of future photoreactors.

Characteristics of Heart Failure Patients With or Without Hypotension When Transitioning From Nitroprusside to Sacubitril-Valsartan.

BACKGROUND: Sacubitril-valsartan is an angiotensin receptor-neprilysin inhibitor indicated for the treatment of patients with symptomatic heart failure with reduced ejection fraction (HFrEF). Little is known about outcomes of HFrEF patients transitioned from sodium nitroprusside (SNP) to sacubitril-valsartan during an admission for acute decompensated heart failure. We sought to describe characteristics of patients initiated on sacubitril-valsartan while receiving SNP and, in particular, those patients who did and did not experience hypotension requiring interruption or discontinuation of sacubitril-valsartan. METHODS: We performed a retrospective case series of adult patients (>18 years) with HFrEF (left ventricular ejection fraction ≤40%) admitted to the University of Michigan cardiac intensive care unit between July 2018 to September 2020 who received sacubitril-valsartan while on SNP. RESULTS: A total of 15 patients with acute decompensated heart failure were initiated on sacubitril-valsartan while on SNP. The mean age was 57 ± 15.9 years. Seven (46.7%) patients experienced hypotension. The patients in the cohort who experienced hypotension were numerically older (60 ± 17 vs. 55 ± 15.5), and the majority were white (86%). Patients with hypotension had a numerically lower left ventricular ejection fraction (13 ± 4.2 vs. 18 ± 8.2) and higher serum creatinine (1.4 ± 0.54 vs. 0.88 ± 0.25). Seven (100%) patients received a diuretic on the day of sacubitril-valsartan initiation in those who experienced hypotension compared with 2 (25%) in those who did not experience hypotension. CONCLUSIONS: In almost half of patients admitted to the cardiac intensive care unit with acutely decompensated HFrEF, significant hypotension was seen when initiating sacubitril-valsartan while on SNP. Future studies should evaluate appropriate patients for this transition and delineate appropriate titration parameters.

A brief period of darkness induces changes in fatty acid biosynthesis towards accumulation of saturated fatty acids in Chlorella vulgaris UMT-M1 at stationary growth phase.

OBJECTIVE: The effects of a brief (3 days) and prolonged (6 days) period of incubation in darkness and light on the biomass content, lipid content and fatty acid profile in Chlorella vulgaris UMT-M1 were determined. RESULTS: Three days of incubation in darkness increased saturated fatty acid (SFA) content from 34.0 to 41.4% but decreased monounsaturated fatty acid (MUFA) content from 36.7 to 29.8%. Palmitic acid (C16:0) content was increased from 23.2 to 28.9%, whereas oleic acid (C18:1) content was reduced from 35.4 to 28.8%. Total oil content was slightly decreased from 20.4 to 18.7% after 3 days of darkness, without a significant reduction in biomass compared to 3 days of incubation in light. Biomass and oil content was highest in cultures incubated for 6 days in light, however the stimulatory and inhibitory effects of darkness (or light) on SFA and MUFA content was no longer present at 6 days of incubation. CONCLUSIONS: Findings from this study suggests that fatty acid composition in C. vulgaris could be modulated to favor either C16:0 or C18:1 by a brief period of either darkness or light incubation, prior to harvesting.

Coronavirus Disease-2019 in Heart Transplant Recipients in Southeastern Michigan: A Case Series.

BACKGROUND: Since coronavirus disease 2019 (COVID-19) was first identified in Wuhan, China, in December 2019, the number of cases has risen exponentially. Clinical characteristics and outcomes among patients with orthotopic heart transplant (OHT) with COVID-19 remain poorly described. METHODS: We performed a retrospective case series of patients with OHT with COVID-19 admitted to 1 of 2 hospitals in Southeastern Michigan between March 21 and April 22, 2020. Clinical data were obtained through review of the electronic medical record. Final date of follow-up was May 7, 2020. Demographic, clinical, laboratory, radiologic, treatment, and mortality data were collected and analyzed. RESULTS: We identified 13 patients with OHT admitted with COVID-19. The mean age of patients was 61 ± 12 years, 100% were black males, and symptoms began 6 ± 4 days before admission. The most common symptoms included subjective fever (92%), shortness of breath (85%), and cough (77%). Six patients (46%) required admission to the intensive care unit. Two patients (15%) died during hospitalization. CONCLUSIONS: Black men may be at increased risk for COVID-19 among patients with OHT. Presenting signs and symptoms in this cohort are similar to those in the general population. Elevated inflammatory markers on presentation appear to be associated with more severe illness.

Clinical Significance of Expression Changes and Promoter Methylation of PLA2R1 in Tissues of Breast Cancer Patients.

Phospholipase A2 receptor 1 (PLA2R1) expression and its role in the initiation and progression of breast cancer are an unresolved issue. PLA2R1 was found to endorse several tumor suppressive responses, including cellular senescence and apoptosis. Previous in vitro studies demonstrated that DNA hypermethylation was highly associated with the epigenetic silencing of PLA2R1 in breast cancer cell lines. Our objective was to study the level of PLA2R1 mRNA expression and the methylation of its promoter in different histological grades and molecular subtypes of breast cancer. We performed bioinformatics analyses on available human breast cancer expression datasets to assess the PLA2R1 mRNA expression. We used qRT-PCR to evaluate the PLA2R1 mRNA expression and its promoter's methylation in breast cancer tissue in comparison to breast fibroadenomas. Our results describe, for the first time, the expression of PLA2R1 and the methylation of its promoter in human breast cancer tissues. A significant downregulation of PLA2R1, together with hypermethylation of the promoter was detected in breast cancers of different histological grades and molecular subtypes when compared to benign breast tissues. PLA2R1 promoter hypermethylation was associated with aggressive subtypes of breast cancer. In conclusion, PLA2R1 promoter hypermethylation is a potentially useful diagnostic and prognostic biomarker and could serve as a possible therapeutic target in breast cancer.

Influence of nitrogen availability on biomass, lipid production, fatty acid profile, and the expression of fatty acid desaturase genes in Messastrum gracile SE-MC4.

In this study, the effects of limited and excess nitrate on biomass, lipid production, and fatty acid profile in Messastrum gracile SE-MC4 were determined. The expression of fatty acid desaturase genes, namely stearoyl-ACP desaturase (SAD), omega-6 fatty acid desaturase (ω-6 FAD), omega-3 fatty acid desaturase isoform 1 (ω-3 FADi1), and omega-3 fatty acid desaturase isoform 2 (ω-3 FADi2) was also assessed. It was found that nitrate limitation generally increased the total oil, α-linolenic acid (C18:3n3) and total polyunsaturated fatty acid (PUFA) contents in M. gracile. The reduction of nitrate concentration from 1.76 to 0.11 mM increased the total oil content significantly from 32.5 to 41.85% (dry weight). Palmitic (C16:0) and oleic (C18:1) acids as the predominant fatty acids in this microalgae constituted between 82 and 87% of the total oil content and were relatively consistent throughout all nitrate concentrations tested. The expression of SAD, ω-6 FAD, and ω-3 FADi2 genes increased under nitrate limitation, especially at 0.11 mM nitrate. The ω-3 FADi1 demonstrated a binary up-regulation pattern of expression under both nitrate-deficient (0.11 mM) and -excess (3.55 mM) conditions. Thus, findings from this study suggested that limited or excess nitrate could be used as part of a cultivation strategy to increase oil and PUFA content following media optimisation and more efficient culture methodology. Data obtained from the expression of desaturase genes would provide valuable insights into their roles under excess and limited nitrate conditions in M. gracile, potentially paving the way for future genetic modifications.

Performance Evaluation of Time-Frequency Distributions for ECG Signal Analysis.

The non-stationary and multi-frequency nature of biomedical signal activities makes the use of time-frequency distributions (TFDs) for analysis inevitable. Time-frequency analysis provides simultaneous interpretations in both time and frequency domain enabling comprehensive explanation, presentation and interpretation of electrocardiogram (ECG) signals. The diversity of TFDs and specific properties for each type show the need to determine the best TFD for ECG analysis. In this study, a performance evaluation of five TFDs in term of ECG abnormality detection is presented. The detection criteria based on extracted features from most important ECG signal components (QRS) to detect normal and abnormal cases. This is achieved by estimating its energy concentration magnitude using the TFDs. The TFDs analyse ECG signals in one-minute interval instead of conventional time domain approach that analyses based on beat or frame containing several beats. The MIT-BIH normal sinus rhythm ECG database total records of 18 long-term ECG sampled at 128 Hz have been analysed. The tested TFDs include Dual-Tree Wavelet Transform, Spectrogram, Pseudo Wigner-Ville, Choi-Williams, and Born-Jordan. Each record is divided into one-minute slots, which is not considered previously, and analysed. The sample periods (slots) are randomly selected ten minutes interval for each record. This result with 99.44% detection accuracy for 15,735 ECG beats shows that Choi-Williams distribution is most reliable to be used for heart problem detection especially in automated systems that provide continuous monitoring for long time duration.

[Femoral hemodialysis catheter-related bacteremia due to Globicatella sanguinis: challenges in species identification]. / Femoral hemodiyaliz kateteri ile iliskili Globicatella sanguinis bakteremisi: tür düzeyinde tanimlamada karsilasilan sorunlar.

In this case, catheter-related bacteremia due to Globicatella sanguinis in a 43 years old female patient undergoing hemodialysis with the diagnosis of diabetic nephropathy was presented and the methods in the laboratory diagnosis of the rare opportunistic pathogen, Globicatella cins, were nvestigated. A set of peripheral blood cultures and simultaneous catheter culture was obtained from the patient in third of May 2016. Biochemical tests, Phoenix (Becton Dickinson, USA) and MicroScan (Beckman Coulter, USA) automated systems and matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF MS) based Microflex MS (Bruker, Daltonics, Germany) and VITEK MS (database v2.0) (bioMérieux, France) systems were used for the identification of the cultured bacteria. Partial 16S rDNA sequencing was done by using specific p8FPL 5'-AGT TTG ATC ATG GCT CAG-3' and p806R 5'-GGA CTA CCA GGG TAT CTA AT-3' primers. Minimal inhibitory concentrations (MICs) for vancomycin, erythromycin, imipenem, cefotaxime and benzypenicillin were determined by agar gradient method. The blood and catheter cultures yielded the same type of bacterial colonies. Alfa-hemolytic, catalase negative colonies observed on blood agar plates after an over night incubation yielded gram-positive cocci on Gram staining. In Sisli Hamidiye Etfal Hospital, the isolate was identifed as G.sulfidifaciens (score value > 2) by Bruker MS system and as G.sanguinis by Phoenix automated system. In Inönü University, the isolate could not be identified by Microscan automated system while VITEK MS named the isolate as 99.9% G.sanguinis and 98.3% G.sulfidifaciens. The 16S rDNA sequencing identifed the isolate as 100% G.sanguinis (GenBank accessionno. KJ680157.1). The MIC values were 0.38 µg/ml, 1.5 µg/ml, 0.38 µg/ml, > 32 µg/ml and 64 µg/ml for vancomycin, eryrthromycin, imipenem, cefotaxime and benzylpenicillin, respectively. The patient was diagnosed as catheter-related bacteremia and vancomycin (1 x 1 g IV/72 h) was used for up to 10 days. No fever and bacterial growth in cultures were present in her control visits. As G.sanguinis is not among the commonly encountered pathogens and due to difficulties in laboratory diagnosis, it may be missedor mis-identified in clinical laboratories. BD Phoenix and Bruker MS data bases lack G.sulfidifaciens and G.sanguinis, respectively, while the Globicatella genus is not present in MicroScan database. The increased number of medical implementations and the increasing number of immunosuppressed patient populations in recenty ears will lead to the emergence of rare bacteria. Increasing the diagnostic power of clinical microbiology laboratories by conventional and molecular methods and renewal of the databases of commercial identification systems by expanding the pathogen spectrum are significantly important for the prevention and control of infections caused by these agents.

Development of pre-breeding technology for root system study and selection of Kihara Afghan wheat landraces (KAWLR) to enhance wheat breeding in the rain-fed region.

To enhance a root trait-based selection program for rain-fed wheat breeding in Afghanistan, we simulated an efficient pre-breeding drought system. Plants were grown in 1 m pipes as control or 2 m pipes to simulate drought conditions soaking ground water up by capillary action supplemented by two different life supporting irrigations from top of the pipes (T1 and T2 droughts). T1 was used for studying genetic diversity in 360 Kihara Afghan wheat landraces (KAWLR). Both drought treatments were used to evaluate root traits in 30 selected genotypes. KAWLR showed large root length variations under T1, categorized as long root (>200 cm; LR), medium root (100-150 cm; MR) and short root (20-100 cm; SR) systems. LR genotypes were more drought resistant in terms of greater plant survivability under T1 and T2 compared with other groups and were capable of adjusting their root biomass partitioning at deepest part of the soil profile. Majority of the LR genotypes originated from predominantly rain-fed provinces, and most of their agronomic traits were strongly correlated with root biomass deep in the soil in response to drought. Three LR genotypes, including the longest root genotype LR-871 (KU7604), are recommended for rain-fed wheat breeding in Afghanistan.