RESUMO
The role of erythropoietin (EPO) has extended beyond hematopoiesis to include cytoprotection, inotropy, and neurogenesis. Extra-renal EPO has been reported for multiple tissue/cell types, but the physiological relevance remains unknown. Although the EPO receptor is expressed by multiple cardiac cell types and human recombinant EPO increases contractility and confers cytoprotection against injury, whether the heart produces physiologically meaningful amounts of EPO in vivo is unclear. We show a distinct circadian rhythm of cardiac EPO mRNA expression in adult mice and increased mRNA expression during embryogenesis, suggesting physiological relevance to cardiac EPO production throughout life. We then generated constitutive, cardiomyocyte-specific EPO knockout mice driven by the Mlc2v promoter (EPOfl/fl:Mlc2v-cre+/-; EPOΔ/Δ-CM). During cardiogenesis, cardiac EPO mRNA expression and cellular proliferation were reduced in EPOΔ/Δ-CM hearts. However, in adult EPOΔ/Δ- CM mice, total heart weight was preserved through increased cardiomyocyte cross-sectional area, indicating the reduced cellular proliferation was compensated for by cellular hypertrophy. Echocardiography revealed no changes in cardiac dimensions, with modest reductions in ejection fraction, stroke volume, and tachycardia, whereas invasive hemodynamics showed increased cardiac contractility and lusitropy. Paradoxically, EPO mRNA expression in the heart was elevated in adult EPOΔ/Δ-CM, along with increased serum EPO protein content and hematocrit. Using RNA fluorescent in situ hybridization, we found that Epo RNA colocalized with endothelial cells in the hearts of adult EPOΔ/Δ-CM mice, identifying the endothelial cells as a cell responsible for the EPO hyper-expression. Collectively, these data identify the first physiological roles for cardiomyocyte-derived EPO. We have established cardiac EPO mRNA expression is a complex interplay of multiple cell types, where loss of embryonic cardiomyocyte EPO production results in hyper-expression from other cells within the adult heart.
Assuntos
Células Endoteliais , Eritropoetina , Animais , Camundongos , Hiperplasia , Hibridização in Situ Fluorescente , Miócitos Cardíacos , RNA , RNA Mensageiro/genéticaRESUMO
KEY POINTS: In the present study, we provide evidence for divergent physiological responses to moderate compared to severe hypoxia, addressing an important knowledge gap related to severity, duration and after-effects of hypoxia encountered in cardiopulmonary situations. The physiological responses to moderate and severe hypoxia were not proportional, linear or concurrent with the time-of-day. Hypoxia elicited severity-dependent physiological responses that either persisted or fluctuated throughout normoxic recovery. The physiological basis for these distinct cardiovascular responses implicates a shift in the sympathovagal set point and probably not molecular changes at the artery resulting from hypoxic stress. ABSTRACT: Hypoxia is both a consequence and cause of many acute and chronic diseases. Severe hypoxia causes hypertension with cardiovascular sequelae; however, the rare studies using moderate severities of hypoxia indicate that it can be beneficial, suggesting that hypoxia may not always be detrimental. Comparisons between studies are difficult because of the varied classifications of hypoxic severities, methods of delivery and use of anaesthetics. Thus, to investigate the long-term effects of moderate hypoxia on cardiovascular health, radiotelemetry was used to obtain in vivo physiological measurements in unanaesthetized mice during 24 h of either moderate (FIO2=0.15) or severe (FIO2=0.09) hypoxia, followed by 72 h of normoxic recovery. Systolic blood pressure was decreased during recovery following moderate hypoxia but increased following severe hypoxia. Moderate and severe hypoxia increased haeme oxygenase-1 expression during recovery, suggesting parity in hypoxic stress at the level of the artery. Severe but not moderate hypoxia increased the low/high frequency ratio of heart rate variability 72 h post-hypoxia, indicating a shift in sympathovagal balance. Moderate hypoxia dampened the amplitude of circadian rhythm, whereas severe disrupted rhythm during the entire insult, with perturbations persisting throughout normoxic recovery. Thus, hypoxic severity differentially regulates circadian blood pressure.
Assuntos
Hipóxia/fisiopatologia , Animais , Pressão Sanguínea , Frequência Cardíaca , Masculino , Camundongos Endogâmicos C57BLRESUMO
Moderate anemia is associated with increased mortality and morbidity, including acute kidney injury (AKI), in surgical patients. A red blood cell (RBC)-specific antibody model was utilized to determine whether moderate subacute anemia could result in tissue hypoxia as a potential mechanism of injury. Cardiovascular and hypoxic cellular responses were measured in transgenic mice capable of expressing hypoxia-inducible factor-1α (HIF-1α)/luciferase activity in vivo. Antibody-mediated anemia was associated with mild intravascular hemolysis (6 h) and splenic RBC sequestration ( day 4), resulting in a nadir hemoglobin concentration of 89 ± 13 g/l on day 4. At this time point, renal tissue oxygen tension (PtO2) was decreased in anemic mice relative to controls (13.1 ± 4.3 vs. 20.8 ± 3.7 mmHg, P < 0.001). Renal tissue hypoxia was associated with an increase in HIF/luciferase expression in vivo ( P = 0.04) and a 20-fold relative increase in renal erythropoietin mRNA transcription ( P < 0.001) but no increase in renal blood flow ( P = 0.67). By contrast, brain PtO2 was maintained in anemic mice relative to controls (22.7 ± 5.2 vs. 23.4 ± 9.8 mmHg, P = 0.59) in part because of an increase in internal carotid artery blood flow (80%, P < 0.001) and preserved cerebrovascular reactivity. Despite these adaptive changes, an increase in brain HIF-dependent mRNA levels was observed (erythropoietin: P < 0.001; heme oxygenase-1: P = 0.01), providing evidence for subtle cerebral tissue hypoxia in anemic mice. These data demonstrate that moderate subacute anemia causes significant renal tissue hypoxia, whereas adaptive cerebrovascular responses limit the degree of cerebral tissue hypoxia. Further studies are required to assess whether hypoxia is a mechanism for acute kidney injury associated with anemia.
Assuntos
Injúria Renal Aguda/sangue , Anemia/sangue , Anticorpos Monoclonais , Encéfalo/irrigação sanguínea , Eritrócitos/metabolismo , Hipóxia Encefálica/sangue , Rim/irrigação sanguínea , Oxigênio/sangue , Injúria Renal Aguda/imunologia , Injúria Renal Aguda/patologia , Injúria Renal Aguda/fisiopatologia , Anemia/imunologia , Anemia/patologia , Anemia/fisiopatologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Circulação Cerebrovascular , Modelos Animais de Doenças , Eritrócitos/imunologia , Eritrócitos/patologia , Eritropoetina/genética , Eritropoetina/metabolismo , Glicoforinas/sangue , Glicoforinas/imunologia , Hemólise , Hipóxia Encefálica/imunologia , Hipóxia Encefálica/patologia , Hipóxia Encefálica/fisiopatologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Rim/metabolismo , Rim/patologia , Masculino , Camundongos Transgênicos , Circulação Renal , Índice de Gravidade de Doença , Baço/metabolismo , Baço/patologia , Regulação para CimaRESUMO
The obesity epidemic is considered one of the most serious public health problems of the modern world. Physical therapy is the most accessible form of treatment; however, compliance is a major obstacle due to exercise intolerance and dyspnea. Respiratory muscle atrophy is a cause of dyspnea, yet little is known of obesity-induced respiratory muscle dysfunction. Our objective was to investigate whether obesity-induced skeletal muscle wasting occurs in the diaphragm, the main skeletal muscle involved in inspiration, using the Zucker diabetic fatty (ZDF) rat. After 14 wk, ZDF rats developed obesity, hyperglycemia, and insulin resistance, compared with lean controls. Hemodynamic analysis revealed ZDF rats have impaired cardiac relaxation (P = 0.001) with elevated end-diastolic pressure (P = 0.006), indicative of diastolic dysfunction. Assessment of diaphragm function revealed weakness (P = 0.0296) in the absence of intrinsic muscle impairment in ZDF rats. Diaphragm morphology revealed increased fibrosis (P < 0.0001), atrophy (P < 0.0001), and reduced myosin heavy-chain content (P < 0.001), compared with lean controls. These changes are accompanied by activation of the myostatin signaling pathway with increased serum myostatin (P = 0.017), increased gene expression (P = 0.030) in the diaphragm and retroperitoneal adipose (P = 0.033), and increased SMAD2 phosphorylation in the diaphragm (P = 0.048). Here, we have confirmed the presence of respiratory muscle atrophy and weakness in an obese, diabetic model. We have also identified a pathological role for myostatin signaling in obesity, with systemic contributions from the adipose tissue, a nonskeletal muscle source. These findings have significant implications for future treatment strategies of exercise intolerance in an obese, diabetic population.
Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Debilidade Muscular/fisiopatologia , Músculos Respiratórios/fisiopatologia , Animais , Diabetes Mellitus Experimental/complicações , Hemodinâmica , Resistência à Insulina , Masculino , Debilidade Muscular/patologia , Miostatina/metabolismo , Obesidade/fisiopatologia , Ratos , Ratos Zucker , Músculos Respiratórios/patologia , Transdução de Sinais , Proteína Smad2/genética , Proteína Smad2/metabolismo , Regulação para CimaRESUMO
The sugary-2 mutation in maize (Zea mays L.) is a result of the loss of catalytic activity of the endosperm-specific SS (starch synthase) IIa isoform causing major alterations to amylopectin architecture. The present study reports a biochemical and molecular analysis of an allelic variant of the sugary-2 mutation expressing a catalytically inactive form of SSIIa and sheds new light on its central role in protein-protein interactions and determination of the starch granule proteome. The mutant SSIIa revealed two amino acid substitutions, one being a highly conserved residue (Gly522âArg) responsible for the loss of catalytic activity and the inability of the mutant SSIIa to bind to starch. Analysis of protein-protein interactions in sugary-2 amyloplasts revealed the same trimeric assembly of soluble SSI, SSIIa and SBE (starch-branching enzyme) IIb found in wild-type amyloplasts, but with greatly reduced activities of SSI and SBEIIb. Chemical cross-linking studies demonstrated that SSIIa is at the core of the complex, interacting with SSI and SBEIIb, which do not interact directly with each other. The sugary-2 mutant starch granules were devoid of amylopectin-synthesizing enzymes, despite the fact that the respective affinities of SSI and SBEIIb from sugary-2 for amylopectin were the same as observed in wild-type. The data support a model whereby granule-bound proteins involved in amylopectin synthesis are partitioned into the starch granule as a result of their association within protein complexes, and that SSIIa plays a crucial role in trafficking SSI and SBEIIb into the granule matrix.
Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/química , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Glucanos/química , Glicogênio Sintase/química , Proteínas de Plantas/química , Sintase do Amido/química , Amido/química , Enzima Ramificadora de 1,4-alfa-Glucana/genética , Alelos , Sequência de Aminoácidos , Amilopectina/química , Glucanos/genética , Glicogênio Sintase/genética , Dados de Sequência Molecular , Proteínas de Plantas/genética , Ligação Proteica/genética , Amido/genética , Sintase do Amido/genética , Zea mays/enzimologiaRESUMO
Type 1 diabetes (T1D) has been reported to negatively affect the health of skeletal muscle, though the underlying mechanisms are unknown. Myostatin, a myokine whose increased expression is associated with muscle-wasting diseases, has not been reported in humans with T1D but has been demonstrated to be elevated in preclinical diabetes models. Thus, the purpose of this study was to determine if there is an elevated expression of myostatin in the serum and skeletal muscle of persons with T1D compared to controls. Secondarily, we aimed to explore relationships between myostatin expression and clinically important metrics (e.g., HbA1c , strength, lean mass) in women and men with (N = 31)/without T1D (N = 24) between 18 and 72 years old. Body composition, baseline strength, blood sample and vastus lateralis muscle biopsy were evaluated. Serum, but not muscle, myostatin expression was significantly elevated in those with T1D versus controls, and to a greater degree in T1D women than T1D men. Serum myostatin levels were not significantly associated with HbA1c nor disease duration. A significant correlation between serum myostatin expression and maximal voluntary contraction (MVC) and body fat mass was demonstrated in control subjects, but these correlations did not reach significance in those with T1D (MVC: R = 0.64 controls vs. R = 0.37 T1D; Body fat: R = -0.52 controls/R = -0.02 T1D). Collectively, serum myostatin was correlated with lean mass (R = 0.45), and while this trend was noted in both groups separately, neither reached statistical significance (R = 0.47 controls/R = 0.33 T1D). Overall, while those with T1D exhibited elevated serum myostatin levels (particularly females) myostatin expression was not correlated with clinically relevant metrics despite some of these relationships existing in controls (e.g., lean/fat mass). Future studies will be needed to fully understand the mechanisms underlying increased myostatin in T1D, with relationships to insulin dosing being particularly important to elucidate.
Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Músculo Esquelético/metabolismo , Miostatina/metabolismo , Adiposidade , Adolescente , Adulto , Idoso , Diabetes Mellitus Tipo 1/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Contração Muscular , Músculo Esquelético/fisiopatologia , Miostatina/sangue , Miostatina/genética , Fatores SexuaisRESUMO
A growing proportion of heart failure (HF) patients present with impairments in both ventricles. Experimental pressure-overload (i.e., transverse aortic constriction, TAC) induces left ventricle (LV) hypertrophy and failure, as well as right ventricle (RV) dysfunction. However, little is known about the coordinated progression of biventricular dysfunction that occurs in TAC. Here we investigated the time course of systolic and diastolic function in both the LV and RV concurrently to improve our understanding of the chronology of events in TAC. Hemodynamic, histological, and morphometric assessments were obtained from the LV and RV at 2, 4, 9, and 18 weeks post-surgery. Results: Systolic pressures peaked in both ventricles at 4 weeks, thereafter steadily declining in the LV, while remaining elevated in the RV. The LV and RV followed different structural and functional timelines, suggesting the patterns in one ventricle are independent from the opposing ventricle. RV hypertrophy/fibrosis and pulmonary arterial remodeling confirmed a progressive right-sided pathology. We further identified both compensation and decompensation in the LV with persistent concentric hypertrophy in both phases. Finally, diastolic impairments in both ventricles manifested as an intricate progression of multiple parameters that were not in agreement until overt systolic failure was evident. Conclusion: We establish pulmonary hypertension was secondary to LV dysfunction, confirming TAC is a model of type II pulmonary hypertension. This study also challenges some common assumptions in experimental HF (e.g., the relationship between fibrosis and filling pressure) while addressing a knowledge gap with respect to temporality of RV remodeling in pressure-overload.
RESUMO
Different methods were used to investigate biofilm growth including crystal violet staining, ATP bioluminescence and total viable count. Seven strains of Listeria monocytogenes and 8 of their derivative strains were screened for their capacity to form biofilms. Both adaptation to benzalkonium chloride (BC) and curing of plasmids did not significantly affect biofilm-forming ability. The strains of L. monocytogenes belonging to serotype 1 formed biofilms significantly better as compared to serotype 4 (P=0.0003). To estimate the efficacy of BC for biofilm elimination the best and the poorest biofilm-formers were used (C719 and LJH 381). It was observed that, L. monocytogenes strain C719 in biofilms is at least 1000 times more resistant to BC than in planktonic form. Cells present in biofilms were shown to recover and grow after BC treatment thus providing a source of recontamination. It was shown that ATP bioluminescence provides good correlation with bacterial counts of L. monocytogenes in biofilms. Staining with crystal violet, on the contrary, did not correlate with bacterial growth in biofilms in the presence of high concentrations of BC but provided information on the concentration of bacterial cells, both live and dead, attached to the surface. ATP bioluminescence was found to be a reliable method for rapid estimation of the efficacy of sanitizers for biofilm disinfection. Crystal violet staining, on the other hand, was shown to be a suitable method to monitor removal of biofilms. Our investigation showed that for Listeria biofilms concentrations of BC higher then 10 mg/ml should be applied for at least 30 min to kill almost all the live cells in biofilms. However, this concentration was still not enough to remove biofilms from the surface of plastic.
Assuntos
Trifosfato de Adenosina/análise , Compostos de Benzalcônio/farmacologia , Biofilmes/efeitos dos fármacos , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/fisiologia , Aderência Bacteriana/fisiologia , Medições Luminescentes/métodos , Coloração e Rotulagem/métodosRESUMO
Diaphragmatic weakness is a feature of heart failure (HF) associated with dyspnea and exertional fatigue. Most studies have focused on advanced stages of HF, leaving the cause unresolved. The long-standing theory is that pulmonary edema imposes a mechanical stress, resulting in diaphragmatic remodeling, but stable HF patients rarely exhibit pulmonary edema. We investigated how diaphragmatic weakness develops in two mouse models of pressure overload-induced HF. As in HF patients, both models had increased eupneic respiratory pressures and ventilatory drive. Despite the absence of pulmonary edema, diaphragmatic strength progressively declined during pressure overload; this decline correlated with a reduction in diaphragm cross-sectional area and preceded evidence of muscle weakness. We uncovered a functional codependence between angiotensin II and ß-adrenergic (ß-ADR) signaling, which increased ventilatory drive. Chronic overdrive was associated with increased PERK (double-stranded RNA-activated protein kinase R-like ER kinase) expression and phosphorylation of EIF2α (eukaryotic translation initiation factor 2α), which inhibits protein synthesis. Inhibition of ß-ADR signaling after application of pressure overload normalized diaphragm strength, Perk expression, EIF2α phosphorylation, and diaphragmatic cross-sectional area. Only drugs that were able to penetrate the blood-brain barrier were effective in treating ventilatory overdrive and preventing diaphragmatic atrophy. These data provide insight into why similar drugs have different benefits on mortality and symptomatology, despite comparable cardiovascular effects.
Assuntos
Insuficiência Cardíaca/terapia , Debilidade Muscular/fisiopatologia , Angiotensina II/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Diafragma/metabolismo , Diafragma/fisiologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Insuficiência Cardíaca/fisiopatologia , Pulmão/metabolismo , Masculino , Camundongos , Debilidade Muscular/metabolismo , Fosforilação/fisiologia , Respiração , Transdução de Sinais/fisiologiaRESUMO
INTRODUCTION: Heme oxygenase-1 (HO-1) is a cytoprotective enzyme induced by stress. Heart failure is a condition of chronic stress-induced remodeling and is often accompanied by comorbidities such as age and hypertension. HO-1 is known to be protective in the setting of acute myocardial infarction. The role of HO-1 in heart failure is not known, particularly in the setting of pressure overload. METHODS: Mice with alpha-myosin heavy chain restricted expression of HO-1 were aged for 1 year. In addition, mice underwent transverse aortic constriction (TAC) or were infused with isoproterenol (ISO) to induce heart failure. RESULTS: HO-1 transgenic mice developed spontaneous heart failure after 1 year compared to their wild-type littermates and showed accelerated cardiac dysfunction 2 weeks following TAC. Wild-type mice undergoing pressure overload demonstrated extensive interstitial fibrosis that was prevented by HO-1 overexpression, yet HO-1 transgenic mice had reduced capillary density, contractile reserve, and elevated end-diastolic pressure. However, HO-1 transgenic mice had significantly attenuated ISO-induced cardiac dysfunction, interstitial fibrosis, and hypertrophy compared to control. Isolated cardiomyocytes from HO-1 transgenic mice treated with ISO did not show evidence of hypercontracture/necrosis and had reduced NADH oxidase activity. CONCLUSIONS: HO-1 is an effective mechanism for reducing acute myocardial stress such as excess beta-adrenergic activity. However, in our age and pressure overload models, HO-1 showed detrimental rather than therapeutic effects in the development of heart failure.
Assuntos
Cardiomiopatias/prevenção & controle , Insuficiência Cardíaca/etiologia , Heme Oxigenase-1/metabolismo , Envelhecimento/patologia , Envelhecimento/fisiologia , Animais , Cardiomiopatias/induzido quimicamente , Cardiomiopatias/patologia , Modelos Animais de Doenças , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Heme Oxigenase-1/genética , Humanos , Hipertensão/complicações , Isoproterenol/toxicidade , Masculino , Camundongos , Camundongos Transgênicos , Miocárdio/enzimologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Regulação para CimaRESUMO
The purpose of the study was to develop a highly sensitive real-time polymerase chain reaction (PCR) assay to detect and quantitate fowl adenovirus (FAdV) DNA in chicken tissues, using FAdV-9 as a model. The assay had a dynamic range of 7 logs and minimum detection limit of 9.4 viral genome copies. It was shown to be highly specific, as tissues from uninfected chickens and other viral genomes, such as those of Marek's disease virus, fowlpox virus and infectious laryngotracheitis virus did not produce positive signal. The sensitivity of the real-time PCR was comparable with nested PCR and it was 100 times more sensitive than the conventional PCR. The assay was validated by testing DNA from tissues of chickens infected with FAdV-9 collected at different days post-infection. FAdV-9 DNA was detected in liver, bursa of Fabricius and cecal tonsil tissues in a range of 10(2)-10(7) copies per 100 ng of total DNA. High amounts of viral DNA were present in the cecal tonsils for a week after inoculation making this tissue an ideal sample source for the diagnosis of FAdV infection. This assay is an excellent research and diagnostic tool that provides high sensitivity, specificity and rapid post-PCR analyses.
Assuntos
Infecções por Adenoviridae , Aviadenovirus/isolamento & purificação , Genoma Viral , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Infecções por Adenoviridae/veterinária , Infecções por Adenoviridae/virologia , Animais , Aviadenovirus/fisiologia , Bolsa de Fabricius/virologia , Galinhas , Replicação do DNA , DNA Viral/isolamento & purificação , Fígado/virologia , Sensibilidade e Especificidade , Replicação ViralRESUMO
Nineteen Listeria monocytogenes strains were characterized by automated ribotyping, pulsed-field gel electrophoresis, and plasmid profiling to determine the relationship between genotype and sanitizer resistance. Isolates within a ribogroup had a consistent sensitivity or resistance phenotype except for ribogroup C isolates. All isolates with resistance phenotypes harbored two plasmids. The sensitivity of L. monocytogenes strains to quaternary ammonium compounds (QACs) was correlated with sensitivity to sanitizers and antibiotics with other modes of action. All isolates tested contained the mdrL gene, which encodes an efflux pump that confers resistance to QACs and is both chromosome and plasmid borne.
Assuntos
Desinfetantes/farmacologia , Listeria monocytogenes/efeitos dos fármacos , Carne/microbiologia , Animais , Farmacorresistência Bacteriana/genética , Indústria de Processamento de Alimentos , Listeria monocytogenes/genética , Plasmídeos , Compostos de Amônio Quaternário/farmacologiaRESUMO
Many studies have demonstrated that bacteria, including Listeria monocytogenes, are capable of adapting to disinfectants used in industrial settings after prolonged exposure to sublethal concentrations. However, the consequent alterations of the cell surface due to sanitizer adaptation of this pathogen are not fully understood. Two resistant and four sensitive L. monocytogenes strains from different sources were progressively subcultured with increasing sublethal concentrations of a surfactant, benzalkonium chloride (BC). To evaluate the effects of acquired tolerance to BC, parent and adapted strains were compared by using several morphological and physiological tests. Sensitive strains showed at least a fivefold increase in the MIC, while the MIC doubled for resistant strains after the adaptation period. The hydrophobicities of cells of parent and adapted strains were similar. Serological testing indicated that antigen types 1 and 4 were both present on the cell surface of adapted cells. The data suggest that efflux pumps are the major mechanism of adaptation in sensitive strains and are less important in originally resistant isolates. A different, unknown mechanism was responsible for the original tolerance of resistant isolates. In an originally resistant strain, there was a slight shift in the fatty acid profile after adaptation, whereas sensitive strains had similar profiles. Electron micrographs revealed morphological differences after adaptation. The changes in cell surface antigens, efflux pump utilization, and fatty acid profiles suggest that different mechanisms are used by resistant and sensitive strains for adaptation to BC.