Exercise alters the circadian rhythm of REV-ERB-α and downregulates autophagy-related genes in peripheral and central tissues.

The transcriptional repressor REV-ERB-α, encoded by Nuclear Receptor Subfamily 1 Group D Member 1 (Nr1d1), has been considered to play an essential role in the skeletal muscle oxidative capacity adaptation and muscle mass control. Also, this molecule regulates autophagy via the repression of autophagy-related genes both in skeletal muscle and brain regions. Classically, training programs based on endurance or strength characteristics enhance skeletal muscle mass content and/or oxidative capacity, leading to autophagy activation in several tissues. Thus, it seems that REV-ERB-α regulates similar responses induced by exercise. However, how this molecule responds to different exercise models/intensities in different tissues is still unclear. Therefore, the main aim was to characterize the responses of REV-ERB-α and autophagy-related genes to different exercise protocols (endurance/interval run/strength) in distinct tissues (gastrocnemius, soleus and hippocampus). Since REV-ERB-α presents a circadian rhythm, the analyses were performed in a time-course manner. The endurance and strength groups attenuated REV-ERB-α transcriptional response during the time course in gastrocnemius and soleus. Conversely, the interval group enhanced the Nr1d1 expression in the hippocampus. All protocols downregulated the REV-ERB-α protein levels in gastrocnemius following the exercise session with concomitant nuclear exclusion. The major autophagy-related genes presented downregulation after the exercise session in all analyzed tissues. Altogether, these results highlight that REV-ERB-α is extremely sensitive to physical exercise stimuli, including different models and intensities in skeletal muscle and the hippocampus.

Molecular evidence for stimulation of methane oxidation in Amazonian floodplains by ammonia-oxidizing communities.

Ammonia oxidation is the rate-limiting first step of nitrification and a key process in the nitrogen cycle that results in the formation of nitrite (NO2 -), which can be further oxidized to nitrate (NO3 -). In the Amazonian floodplains, soils are subjected to extended seasons of flooding during the rainy season, in which they can become anoxic and produce a significant amount of methane (CH4). Various microorganisms in this anoxic environment can couple the reduction of different ions, such as NO2 - and NO3 -, with the oxidation of CH4 for energy production and effectively link the carbon and nitrogen cycle. Here, we addressed the composition of ammonium (NH4 +) and NO3 --and NO2 --dependent CH4-oxidizing microbial communities in an Amazonian floodplain. In addition, we analyzed the influence of environmental and geochemical factors on these microbial communities. Soil samples were collected from different layers of forest and agroforest land-use systems during the flood and non-flood seasons in the floodplain of the Tocantins River, and next-generation sequencing of archaeal and bacterial 16S rRNA amplicons was performed, coupled with chemical characterization of the soils. We found that ammonia-oxidizing archaea (AOA) were more abundant than ammonia-oxidizing bacteria (AOB) during both flood and non-flood seasons. Nitrogen-dependent anaerobic methane oxidizers (N-DAMO) from both the archaeal and bacterial domains were also found in both seasons, with higher abundance in the flood season. The different seasons, land uses, and depths analyzed had a significant influence on the soil chemical factors and also affected the abundance and composition of AOA, AOB, and N-DAMO. During the flood season, there was a significant correlation between ammonia oxidizers and N-DAMO, indicating the possible role of these oxidizers in providing oxidized nitrogen species for methanotrophy under anaerobic conditions, which is essential for nitrogen removal in these soils.

Structural basis for effector recognition by an antibacterial type IV secretion system.

Many soil-, water-, and plant-associated bacterial species from the orders Xanthomonadales, Burkholderales, and Neisseriales carry a type IV secretion system (T4SS) specialized in translocating effector proteins into other gram-negative species, leading to target cell death. These effectors, known as X-Tfes, carry a carboxyl-terminal domain of ∼120 residues, termed XVIPCD, characterized by several conserved motifs and a glutamine-rich tail. Previous studies showed that the XVIPCD is required for interaction with the T4SS coupling protein VirD4 and for T4SS-dependent translocation. However, the structural basis of the XVIPCD-VirD4 interaction is unknown. Here, we show that the XVIPCD interacts with the central all-alpha domain of VirD4 (VirD4AAD). We used solution NMR spectroscopy to solve the structure of the XVIPCD of X-TfeXAC2609 from Xanthomonas citri and to map its interaction surface with VirD4AAD Isothermal titration calorimetry and in vivo Xanthomonas citri versus Escherichia coli competition assays using wild-type and mutant X-TfeXAC2609 and X-TfeXAC3634 indicate that XVIPCDs can be divided into two regions with distinct functions: the well-folded N-terminal region contains specific conserved motifs that are responsible for interactions with VirD4AAD, while both N- and carboxyl-terminal regions are required for effective X-Tfe translocation into the target cell. The conformational stability of the N-terminal region is reduced at and below pH 7.0, a property that may facilitate X-Tfe unfolding and translocation through the more acidic environment of the periplasm.

Histo-blood group A is a risk factor for severe COVID-19.

OBJECTIVES: Evaluate the impact of ABO histo-blood group type on COVID-19 severity. BACKGROUND: ABO histo-blood type has been associated with different outcomes in infectious diseases. It has also shown a higher proportion of type A patients with SARS-CoV-2. In this observational study, extracted from an ongoing clinical trial on the efficacy of convalescent plasma transfused in COVID-19 patients, we describe the impact of ABO blood type on the risk of developing severe COVID-19. MATERIALS AND METHODS: Seventy-two consecutive patients (37 type A, 23 type O, 11 type B, 1 type AB) with severe (respiratory failure) COVID-19 were included. Control group was composed of 160 individuals randomly selected from the same populational basis. RESULTS: Blood group A was overrepresented (51.39%) in the patient group in relation to the control group (30%), whereas blood group O was less represented (31.94%) in patient than in control group (48%). Odds ratio (A vs. O) was 2.581 (1.381-4.817), CI 95%; p = 0.004. Also, blood group A patients appeared to have more severe disease, given by the scores of the Sequential Organ Failure Assessment and Simplified Acute Physiologic Score 3 (p = 0.036 and p = 0.058, respectively). CONCLUSION: Histo-blood type A is associated with a higher risk of developing severe COVID-19 in relation to blood type O.

Methane emission suppression in flooded soil from Amazonia.

The coupling between ferrous iron and methane production has important global implications, with iron ions acting as electron acceptors for anaerobic oxidation of methane (AOM) and inhibitors of methanogenesis in different environments, including floodplain soils. In this sense, we analyzed the relationship between Fe(II) concentration and methane production in soil layers collected at 0-15 cm and 15-30 cm from flooded-forest and -agroforestry in Amazonian clear water floodplain incubated in anaerobic batch reactors using acetate, formate and glucose as organic sources. High throughput sequencing of archaeal and bacterial 16S rRNA genes was employed to assess the abundance and composition of the active methanogenic and methanotrophic microbial groups potentially involved in Fe(III)-dependent AOM in the soil used as inoculum. Positive correlation was revealed between Fe(II) concentration and methane production, with higher accumulation of Fe(II) in incubated soil layer collected at 0-15 cm in both forest and agroforestry sites for all the three organic sources. The accumulation of Fe(II) in the incubated soil evidenced the oxidation of Fe(III) potentially by Methanobacterium, Desulfobulbus and 'Candidatus methanoperedens nitroreducens' living in anaerobic condition at this soil layer. The results point out to the microbial ferric iron reduction as an important potential pathway for anaerobic organic matter decomposition in Amazonian floodplain, evidencing methanogenesis suppression by Fe(III) reduction in flooded-forest and -agroforestry in Amazonian clear water river floodplain.

Effect of the levonorgestrel-releasing intrauterine system on cardiovascular risk markers among women with thrombophilia or previous venous thromboembolism.

OBJECTIVE: To assess the effects of the levonorgestrel-releasing intrauterine system (LNG-IUS) on standard cardiovascular risk markers among women with thrombophilia and/or previous venous thromboembolism (VTE). METHODS: A prospective cohort study enrolled women aged 18-45 years with thrombophilia and/or a history of VTE who received the 52-mg LNG-IUS (20 µg/d initial release) at the University of Ribeirão Preto Medical School, Brazil, from January 2006 to December 2015. Before and 12 months after LNG-IUS placement, the following cardiovascular risk markers were assessed: lipid profile, body mass index (BMI), blood glucose, systolic blood pressure, diastolic blood pressure, and waist circumference. The primary outcome was changes in cardiovascular risk markers. A subanalysis of anticoagulant users versus non-users was also conducted. RESULTS: In total, 45 women were enrolled. BMI increased by 2.3% after 12 months of LNG-IUS placement (P < 0.01), but the other risk factors did not change. Cardiovascular risk markers were similar between anticoagulant users and non-users after 12 months of LNG-IUS use. CONCLUSION: Among women with thrombophilia and/or previous VTE, cardiovascular risk markers were not found to change significantly after 12 months of LNG-IUS use. The study adds safety information regarding use of the LNG-IUS for women at risk of thromboembolism.

Observational study of multiple myeloma in Latin America.

Relatively little is known about the outcomes of multiple myeloma in Latin America, a world region where incorporation of novel agents is generally slow. In the current retrospective-prospective study, we aimed to describe the patterns of care and treatment results in five Latin American countries. Between April 2007 and October 2009, patients who had been diagnosed from January 2005 to December 2007 were registered at 23 institutions from Argentina, Brazil, Chile, Mexico, and Peru. We divided patients into two cohorts, according to transplantation eligibility, and analyzed them with regard to first-line treatment and overall survival (OS). We analyzed a total of 852 patients, 46.9 % of whom were female. The median follow-up was 62 months. Among transplantation-ineligible patients (N = 461), the mean age was 67.4 years, approximately one third of patients received a thalidomide-based treatment in the first line, and the median OS was 43.0 months. Transplantation-eligible patients (N = 391) had a mean age of 54.7 years and a median OS of 73.6 months. Autologous transplantation was performed in 58.6 % of the patients for whom this procedure was initially planned and in only 26.9 % of the overall patients. Our long-term results reflect the contemporary literature for patients with multiple myeloma treated with autologous transplantation and thalidomide-based regimens in clinical trials and observational studies. However, further efforts are needed to approve and incorporate novel agents in Latin American countries, as well as to increase access to transplantation, in order to achieve the expected improvements in patient outcomes.

Exhaustive Training Leads to Hepatic Fat Accumulation.

Recently, we demonstrated that an overtraining (OT) protocol for mice based on downhill running sessions increased the hepatic phosphorylation of 70-kDa ribosomal protein S6 kinase 1 (S6K1; Thr389), a downstream target of the mammalian target of rapamycin complex 1 (mTORC1). In liver, the overactivation of the Akt/mTORC1 pathway induces lipogenesis via regulation of the action of sterol regulatory element binding protein-1 (SREBP-1) at multiple steps. Herein, we verified the effects of three running OT models with same external load (i.e., the product between intensity and volume of training), but performed in downhill, uphill and without inclination, on the proteins related to the mTORC1 signaling pathway, the protein content of the SREBP-1, ACC, and FAS, and the morphological characteristics of C57BL/6 mouse livers. In summary, the downhill running-induced OT model up-regulated the levels of major proteins of the mTORC1 signaling pathway, the protein levels of SREBP-1 (p125 precursor) and induced signs of cell swelling accompanied by acute inflammation. The other two OT protocols performed uphill and without inclination did not modulate the most analyzed molecular proteins, but induced hepatic morphological alterations, suggesting an acute pathological adaptation. The three OT models induced hepatic fat accumulation. J. Cell. Physiol. 232: 2094-2103, 2017. © 2016 Wiley Periodicals, Inc.

Increased expression of miR-221 is associated with shorter overall survival in T-cell acute lymphoid leukemia.

BACKGROUND: CD56 expression has been associated with a poor prognosis in lymphoid neoplasms, including T-cell acute lymphoblastic leukemia (T-ALL). MicroRNAs (miRNAs) play an important role in lymphoid differentiation, and aberrant miRNA expression has been associated with treatment outcome in lymphoid malignancies. Here, we evaluated miRNA expression profiles in normal thymocytes, mature T-cells, and T-ALL samples with and without CD56 expression and correlated microRNA expression with treatment outcome. METHODS: The gene expression profile of 164 miRNAs were compared for T-ALL/CD56+ (n=12) and T-ALL/CD56- (n=36) patients by Real-Time Quantitative PCR. Based on this analysis, we decided to evaluate miR-221 and miR-374 expression in individual leukemic and normal samples. RESULTS: miR-221 and miR-374 were expressed at significantly higher levels in T-ALL/CD56+ than in T-ALL/CD56- cells and in leukemic blasts compared with normal thymocytes and peripheral blood (PB) T-cells. Age at diagnosis (15 or less vs grater than 15 years; HR: 2.19, 95% CI: 0.98-4.85; P=0.05), miR-221 expression level (median value as cut off in leukemic samples; HR: 3.17, 95% CI: 1.45-6.92; P=0.004), and the expression of CD56 (CD56-vs CD56+; HR: 2.99, 95% CI: 1.37-6.51; P=0.006) were predictive factors for shorter overall survival; whereas, only CD56 expression (HR: 2.73, 95% CI: 1.03-7.18; P=0.041) was associated with a shorter disease-free survival rate. CONCLUSIONS: miR-221 is highly expressed in T-ALL and its expression level may be associated with a poorer prognosis.

Thalidomide plus dexamethasone as a maintenance therapy after autologous hematopoietic stem cell transplantation improves progression-free survival in multiple myeloma.

Despite the good response of stem cell transplant (SCT) in the treatment of multiple myeloma (MM), most patients relapse or do not achieve complete remission, suggesting that additional treatment is needed. We assessed the impact of thalidomide in maintenance after SCT in untreated patients with MM. A hundred and eight patients (<70 years old) were randomized to receive maintenance with dexamethasone (arm A; n = 52) or dexamethasone with thalidomide (arm B; n = 56; 200 mg daily) for 12 months or until disease progression. After a median follow-up of 27 months, an intention to treat analysis showed a 2-year progression-free survival (PFS) of 30% in arm A (95% CI 22-38) and 64% in arm B (95% CI 57-71; P = 0.002), with median PFS of 19 months and 36 months, respectively. In patients who did not achieve at least a very good partial response, the PFS at 2 years was significantly higher when in use of thalidomide (19 vs. 59%; P = 0.002). Overall survival at 2 years was not significantly improved (70 vs. 85% in arm A and arm B, respectively; P = 0.27). The addition of thalidomide to dexamethasone as maintenance improved the PFS mainly in patients who did not respond to treatment after SCT.

The binding protein BiP attenuates stress-induced cell death in soybean via modulation of the N-rich protein-mediated signaling pathway.

The molecular chaperone binding protein (BiP) participates in the constitutive function of the endoplasmic reticulum (ER) and protects the cell against stresses. In this study, we investigated the underlying mechanism by which BiP protects plant cells from stress-induced cell death. We found that enhanced expression of BiP in soybean (Glycine max) attenuated ER stress- and osmotic stress-mediated cell death. Ectopic expression of BiP in transgenic lines attenuated the leaf necrotic lesions that are caused by the ER stress inducer tunicamycin and also maintained shoot turgidity upon polyethylene glycol-induced dehydration. BiP-mediated attenuation of stress-induced cell death was confirmed by the decreased percentage of dead cell, the reduced induction of the senescence-associated marker gene GmCystP, and reduced DNA fragmentation in BiP-overexpressing lines. These phenotypes were accompanied by a delay in the induction of the cell death marker genes N-RICH PROTEIN-A (NRP-A), NRP-B, and GmNAC6, which are involved in transducing a cell death signal generated by ER stress and osmotic stress through the NRP-mediated signaling pathway. The prosurvival effect of BiP was associated with modulation of the ER stress- and osmotic stress-induced NRP-mediated cell death signaling, as determined in transgenic tobacco (Nicotiana tabacum) lines with enhanced (sense) and suppressed (antisense) BiP levels. Enhanced expression of BiP prevented NRP- and NAC6-mediated chlorosis and the appearance of senescence-associated markers, whereas silencing of endogenous BiP accelerated the onset of leaf senescence mediated by NRPs and GmNAC6. Collectively, these results implicate BiP as a negative regulator of the stress-induced NRP-mediated cell death response.

Influence of age on reactivity to diverse emotional challenges in low- and high-anxiety rats.

Studies have revealed that the extent of reactivity of high-anxiety rats to diverse challenges is different than low-anxiety rats and have provided important insights into the psychopathology of anxiety. Various factors intervene to allow defensive mechanisms to react to diverse threatening challenges, including ontogeny and the nature of the emotional challenge (e.g., conditioned vs. unconditioned). The present study investigated the extent to which a particular type of fear extrapolates to other emotional responses to diverse threatening challenges. Groups of 30- and 60-day-old rats were assigned to low freezing behavior (LFB) and high freezing behavior (HFB) groups using the contextual fear conditioning paradigm and subjected to either the fear-potentiated startle (FPS) test, novelty-induced ultrasound vocalizations (USVs) or elevated plus-maze (EPM) tests. At 30 days of age, HFB rats exhibited greater FPS than LFB rats. In contrast, prior selection of HFB and LFB did not affect the performance of 30-day-old animals in the EPM and novelty-induced USVs. Sixty-day-old animals exhibited a performance deficit in all three tests. These data suggest that the performance of young rats in animal models of anxiety parallels their selection as LFB and HFB in the contextual fear conditioning paradigm. However, the increased fear-like behavior exhibited by the 60-day-old HFB rats may elicit performance deficits in conditioned and unconditioned fear tests. These results suggest that the interaction between hyperanxiety and age may cause a performance deficit despite the animals' increased fear-like behavior when facing emotional challenges, thus resembling psychiatric patients in many respects.

The effects of Tynnanthus fasciculatus (Bignoniaceae) infusion on testicular parenchyma of adult Wistar rats.

Traditional medicine provides strong guidance for scientific experiments involving plant products used by the Brazilian people. The species "cipó-cravo" (Tynnanthus fasciculatus) is a plant commonly used either to combat indigestion and stomach aches, or as a general stimulant and aphrodisiac. In this study, the effects of "cipó-cravo" infusion were investigated within the testicular parenchyma of adult Wistar rats. Rats were divided into 3 groups: a control (distilled water) and two treated groups, which received the plant infusion (100 and 200mg/animal/day). The 200mg dose promoted a significant increase of the testicular parenchyma weight and of the volume and total length of the seminiferous tubules, as well as in total daily sperm production and sperm production per gram of testis.

The effects of Tynnanthus fasciculatus (Bignoniaceae) infusion on testicular parenchyma of adult Wistar rats

Traditional medicine provides strong guidance for scientific experiments involving plant products used by the Brazilian people. The species "cipó-cravo" (Tynnanthus fasciculatus) is a plant commonly used either to combat indigestion and stomachaches, or as a general stimulant and aphrodisiac. In this study, the effects of "cipó-cravo" infusion were investigated within the testicular parenchyma of adult Wistar rats. Rats were divided into 3 groups: a control (distilled water) and two treated groups, which received the plant infusion (100 and 200mg/animal/day). The 200mg dose promoted a significant increase of the testicular parenchyma weight and of the volume and total length of the seminiferous tubules, as well as in total daily sperm production and sperm production per gram of testis.

[The psychological wellbeing of health personnel working in public hospitals]. / Saúde Psíquica dos Profissionais de Saúde em Hospitais Públicos.

OBJECTIVES: Analysing the level of psychic health in health workers from public hospitals in the State of Rio Grande do Norte, Brazil. MATERIALS AND METHODS: This was a descriptive, traverse cohort (stratified proportional) study of a sample of 95 health workers; a socio-demographic and work-based questionnaire and the Goldberg's General Health Questionnaire (GGHQ) were used. RESULTS: 82% of the subjects were female, 60% were married, 81% catholic, 48% were aged more than 45, 73% had a workload of 40 hours per week and 63% worked at other places. 64% presented a good level of overall psychic health, the psychic health of 31% of them was becoming affected and 5% were in extreme danger. CONCLUSIONS: The need for preventative action should be stressed, even with psychically healthy professionals, being, to avoid future damage or more serious worsening of these workers' psychic health.

Saúde psíquica dos profissionais de saúde em hospitais públicos / La salud psíquica en los profesionales de la salud en hospitales públicos / The psychic wellbeing of health personnel working in public hospitals

Objetivo Analisar o nível de saúde psíquica dos profissionais de saúde nas enfermarias dos hospitais públicos do Estado do Rio Grande do Norte, Brasil. Materiais e Métodos Foi realizado um estudo descritivo transversal com 95 profissionais de saúde (amostra estratificada proporcional). Utilizou-se um questionário constando características sócio-demográficas e de trabalho, assim como o Questionário de Saúde Geral de Goldberg (QSG). Resultados Os empregados são do sexo feminino, casados, de idade acima de 45 anos. Contam com uma carga horária de trabalho de 40 horas semanais, além de apresentar múltiplos vínculos de trabalho. A saúde psíquica global dos sujeitos encontrase em um nível bom, no entanto, também há indivíduos em número significativo de processo de agravamento, quanto ao stress (37,9 por cento) e distúrbios psicossomáticos (34,7 por cento) isoladamente. Conclusões Há um indicativo de alerta quanto à necessidade de ações preventivas, de modo a evitar maiores agravamentos com possíveis danos às condições de saúde psíquica destes trabalhadores.

Objectives Analysing the level of psychic health in health workers from public hospitals in the State of Rio Grande do Norte, Brazil. Materials and Methods This was a descriptive, traverse cohort (stratified proportional) study of a sample of 95 health workers; a socio-demographic and work-based questionnaire and the Goldberg's General Health Questionnaire (GGHQ) were used. Results 82 percent of the subjects were female, 60 percent were married, 81 percent catholic, 48 percent were aged more than 45, 73 percent had a workload of 40 hours per week and 63 percent worked at other places. 64 percent presented a good level of overall psychic health, the psychic health of 31 percent of them was becoming affected and 5 percent were in extreme danger. Conclusions The need for preventative action should be stressed, even with psychically healthy professionals, being, to avoid future damage or more serious worsening of these workers' psychic health.

Objetivo Analizar el nivel de la salud psíquica de los profesionales de salud en las enfermerías de los hospitales del Estado de Rio Grande do Norte, Brasil. Material y Métodos Fue realizado un estudio descriptivo transversal con 95 profesionales de salud (muestra estratificada proporcional). Se utilizó un cuestionario constando las características socio-demográficas y laborales, así como el Cuestionario de Salud General de Goldberg (QSG). Resultados Los empleados son del sexo femenino, casadas, con edad superior a 45 años. Cuentan con una carga horaria laboral de 40 horas semanales, además de presentar múltiples vínculos de trabajo. La salud psíquica global de los sujetos se encuentra en un nivel bueno, pero también hay individuos en número significativo de procesos de agravamiento, en relación al estrés (37,9 por ciento) y a los disturbios psicosomáticos (34,7 por ciento) independientemente. Conclusiones Hay un indicativo de alerta sobre la necesidad de acciones preventivas,de modo a evitar mayores complicaciones con posibles daños a las condiciones desalud psíquica de estos trabajadores.

Determination of P-glycoprotein, MDR-related protein 1, breast cancer resistance protein, and lung-resistance protein expression in leukemic stem cells of acute myeloid leukemia.

BACKGROUND: The most primitive leukemic precursor in acute myeloid leukemia (AML) is thought to be the leukemic stem cell (LSC), which retains the properties of self-renewal and high proliferative capacity and quiescence of the hematopoietic stem cell. LSC seems to be immunophenotypically distinct and more resistant to chemotherapy than the more committed blasts. Considering that the multidrug resistance (MDR) constitutive expression may be a barrier to therapy in AML, we have investigated whether various MDR transporters were differentially expressed at the protein level by different leukemic subsets. METHODS: The relative expression of the drug-efflux pumps P-gp, MRP, LRP, and BCRP was evaluated by mean fluorescence index (MFI) and the Kolmogorov-Smirnov analysis (D values) in five leukemic subpopulations: CD34+CD38-CD123+ (LSCs), CD34+CD38+CD123-, CD34+CD38+CD123+, CD34+CD38+CD123-, and CD34- mature cells in 26 bone marrow samples of CD34+ AML cases. RESULTS: : The comparison between the two more immature subsets (LSC versus CD34+CD38-CD123- cells) revealed a higher P-gp, MRP, and LRP expression in LSCs. The comparative analysis between LSCs and subsets of intermediate maturation (CD34+CD38+) demonstrated the higher BCRP expression in the LSCs. In addition, P-gp expression was also significantly higher in the LSC compared to CD34+CD38+CD123- subpopulation. Finally, the comparative analysis between LSC and the most mature subset (CD34-) revealed higher MRP and LRP and lower P-gp expression in the LSCs. CONCLUSIONS: Considering the cellular heterogeneity of AML, the higher MDR transporters expression at the most immature, self-renewable, and quiescent LSC population reinforces that MDR is one of the mechanisms responsible for treatment failure.