Prediction of the Impact of CYP2C19 Polymorphism on Drug-Drug Interaction between Voriconazole and Tacrolimus Using Physiologically-Based Pharmacokinetic Modelling

Abstract Voriconazole increases tacrolimus blood concentration significantly when coadministrated. The recommendation of reducing tacrolimus to 1/3 in voriconazole package insert seems not to be satisfactory in clinical practice. In vitro studies demonstrated that the magnitude of inhibition depends on the concentration of voriconazole, while voriconazole exposure is determined by the genotype status of CYP2C19. CYP2C19 gene polymorphism challenges the management of drug-drug interactions(DDIs) between voriconazole and tacrolimus. This work aimed to predict the impact of CYP2C19 polymorphism on the DDIs by using physiologically based pharmacokinetics (PBPK) models. The precision of the developed voriconazole and tacrolimus models was reasonable by evaluating the pharmacokinetic parameters fold error, such as AUC0-24, Cmax and tmax. Voriconazole increased tacrolimus concentration immediately in all population. The simulated duration of DDIs disappearance after voriconazole withdrawal were 146h, 90h and 66h in poor metabolizers (PMs), intermediate metabolizers (IMs) and extensive metabolizers(EMs), respectively. The developed and optimized PBPK models in this study can be applied to assit the dose adjustment for tacrolimus with and without voriconazole.

Risk assessment of internal mammary lymph node metastasis and choice of irradiation of internal mammary lymphatic drainage area in breast cancer patients with negative internal breast lymph nodes on imaging / 中华肿瘤杂志

Objective: To explore the independent risk factors of internal mammary lymph nodes (IMN) metastasis and the risk assessment method of IMN metastasis preoperatively in breast cancer patients with negative IMN in imaging examination, and guide the radiotherapy of IMN in patients with different risk stratification of IMN metastasis. Methods: The clinical and pathological data of 301 breast cancer patients who underwent internal mammary sentinel node biopsy(IM-SLNB) and/or IMN dissection in Shandong Cancer Hospital with negative IMN on CT and/or MRI from January 2010 to October 2019 were analyzed retrospectively. The independent risk factors were analyzed by univariate and multivariate logistic regression, and the independent risk factors of IMN metastasis were used to risk stratification. Results: Among the 301 patients, 43 patients had IMN metastasis, and the rate of IMN metastasis was 14.3%. Univariate analysis showed that vascular tumor thrombus, progesterone receptor (PR) expression, T stage and N stage were associated with IMN metastasis. Multivariate logistic regression analysis showed that tumor located in medial quadrant, positive PR and axillary lymph node metastasis were independent risk factors for IMN metastasis. The risk of IMN metastasis was assessed according to the independent risk factors of the patients: low-risk group is including 0 risk factor, medium-risk group is including 1 risk factor, and high-risk group is including 2-3 risk factors. According to this evaluation criteria, 301 patients with breast cancer were divided into low-risk group (with 0 risk factors), medium-risk group (with 1 risk factor) and high-risk group (with 2-3 risk factors). The IMN metastasis rates were 0 (0/34), 4.3% (6/140) and 29.1% (37/127), respectively. Conclusions: The risk stratification of IMN metastasis according to three independent risk factors of IMN metastasis including tumor located in medial quadrant, positive PR and axillary lymph node metastasis in breast cancer patients can guide the radiotherapy of IMN in newly diagnosed breast cancer patients. For N1 patients, radiotherapy of IMN is strongly recommended when the primary tumor is located in the medial quadrant and/or PR positive.

Recent progress of nanoparticle-enabled modulation of macrophages for cancer immunotherapy / 药学学报

Macrophages play an important role in maintaining homeostasis of the body, and they are also one of the most abundant immune cells in the tumor microenvironment (TME). These macrophages are often called tumor-associated macrophages (TAMs), which play an important role in the development of tumor and are an important target for tumor therapy. Studies have shown that tumor growth and metastasis can be inhibited by regulating the function of macrophages, but the therapeutic efficacy was often hampered by the poor performance of the drugs such as lack of targeting, poor solubility, low bioavailability, and severe side effects. After introduction of the background of macrophage and tumor therapy, this review focuses on the research progress of nano-drug delivery systems in the modulation of the function of macrophages to enhance tumor immunotherapy. Nano-drug delivery systems are diverse in structures and functions, and can regulate macrophage functions through a variety of mechanisms. Four important aspects of macrophage modulation, which included TAMs depletion, repolarization of TAMs, promoted phagocytosis of TAMs, and combinational modulation of TAMs were summarized. Each strategy together with typical examples was reviewed and future directions in this field were also prospected.

Effect of Qingfei Jiangmai Decoction on Content of PM2.5-related Metabolites Mercapturic Acids in Urine / 中国实验方剂学杂志

ObjectiveTo explore the effect of Qingfei Jiangmai decoction （QJD） on the content of mercapturic acids in urine in healthy people amid PM2.5 （particles 2.5 microns or less in size） pollution. MethodA total of 84 healthy students of 18-30 years old in Beijing were recruited and they were randomized into the test group （42 in total， with 1 dropout） and control group （42 in total， with 3 dropouts）. During the pollution， the test group and the control group respectively took QJD granules and placebo for 7 days （1 bag/time， 2 times/day）， and another 7-day intervention with the same drugs was performed at an interval of 4 weeks. The time-activity patterns were recorded during the intervention. On-line solid phase extraction-liquid chromatography/tandem mass spectrometry （SPE-LC-MS/MS） was performed to detect the content of PM2.5-related metabolites S-phenylmercapturic acid （SPMA）， 3-hydroxypropylmercapturic acid （3-HPMA）， 3-hydroxy-1-methylpropylmercapturic acid （HMPMA）， N-acetyl-S-（2-nitrile ethyl）-L-cysteine （CEMA）， and N-acetyl-S-（2-hydroxy ethyl）-L-cysteine （HEMA） in urine before and after intervention. Statistical analysis was followed. ResultThe content of CEMA， HEMA， 3-HPMA， and HMPMA in the test group was all higher after the intervention than before the intervention， with the significant difference in HEMA （P<0.05）. After intervention， content of HEMA and SPMA was significantly higher in the test group than in the control group （P<0.05）， and the difference in HEMA （Z=-3.614， P<0.01） and HMPMA （Z=-1.988， P<0.05） before and after invention in the test group was significantly larger than that in the control group. After the intervention， HEMA in the test group was significantly higher than that in the control group （F=7.597， P<0.01）. ConclusionDuring PM2.5 pollution， QJD can increase the excretion of HEMA， a metabolite of ethylene oxide， in the urine of healthy people in Beijing， and enhance the detoxification process of toxic components in PM2.5， which is of great value in preventing and treating haze-related illnesses.

Incidence of extrauterine growth retardation and its risk factors in very preterm infants during hospitalization: a multicenter prospective study / 中国当代儿科杂志

OBJECTIVES@#To investigate the incidence of extrauterine growth retardation (EUGR) and its risk factors in very preterm infants (VPIs) during hospitalization in China.@*METHODS@#A prospective multicenter study was performed on the medical data of 2 514 VPIs who were hospitalized in the department of neonatology in 28 hospitals from 7 areas of China between September 2019 and December 2020. According to the presence or absence of EUGR based on the evaluation of body weight at the corrected gestational age of 36 weeks or at discharge, the VPIs were classified to two groups: EUGR group (n=1 189) and non-EUGR (n=1 325). The clinical features were compared between the two groups, and the incidence of EUGR and risk factors for EUGR were examined.@*RESULTS@#The incidence of EUGR was 47.30% (1 189/2 514) evaluated by weight. The multivariate logistic regression analysis showed that higher weight growth velocity after regaining birth weight and higher cumulative calorie intake during the first week of hospitalization were protective factors against EUGR (P<0.05), while small-for-gestational-age birth, prolonged time to the initiation of total enteral feeding, prolonged cumulative fasting time, lower breast milk intake before starting human milk fortifiers, prolonged time to the initiation of full fortified feeding, and moderate-to-severe bronchopulmonary dysplasia were risk factors for EUGR (P<0.05).@*CONCLUSIONS@#It is crucial to reduce the incidence of EUGR by achieving total enteral feeding as early as possible, strengthening breastfeeding, increasing calorie intake in the first week after birth, improving the velocity of weight gain, and preventing moderate-severe bronchopulmonary dysplasia in VPIs.

Clinical and imaging characteristics of patients with primary cardiac angiosarcoma / 中华心血管病杂志

Objective: To investigate the clinical, cardiac imaging characteristics and prognosis of patients with primary cardiac angiosarcoma. Methods: The clinical data of 14 patients hospitalized with primary cardiac angiosarcoma from January 2001 to December 2017 in Peking Union Medical College Hospital were collected and analyzed. Metastatic cardiac angiosarcoma was not included in this study. Patients were followed up post discharge per telephone call or clinical visit. Results: Of the 14 patients, 8 were males and 6 were females, average age was 48 years. The main clinical symptoms were shortness of breath (8/14), hemoptysis (6/14), fever (5/14), chest pain (4/14) and cough (3/14). Imaging examinations showed that the tumors of 8 patients were located in the right heart and 6 in the pericardial cavity. Tumors in the right heart often infiltrate the atrial wall and cause pericardial effusion (7/8). Tumors in the pericardium were characterized by recurrent bloody pericardial effusion (6/6), prone to progressive constrictive pericarditis (3/6), pericardial fluid cytology was often negative (6/6). MRI showed heterogeneous high signal intensity (cauliflower aspect) on T2-weighted image and heterogeneous enhancement with a"sunray" aspect at the perfusion study. At the time of diagnosis, 8 patients developed lung or adrenal metastasis (8/14). The median survival was only 305 days. Conclusions: Primary cardiac angiosarcoma is a rare disease with non-specific clinical manifestation and poor prognosis. Imaging examinations may help diagnosis. The high invasiveness and the easy-to-metastasis feature of the tumor contribute to the poor prognosis of cardiac angiosarcoma.

Protective effects of proanthocyanidins from Rubus amabilis Focke on pancreatic tissues of diabetic mice / 中国药理学通报

Aim To study the protective effect of proanthocyanidins extracted from Rubus amabilis Focke (RPC) on the pancreatic tissues of diabetic mice and the underlying mechanism. Methods C57BL/6 male mice were given a high-sugar and high-fat diet combined with an intraperitoneal injection of STZ to establish the diabetic model. The mice whose FBG were higher than 16. 7 mmol · L

Preparation of chrysin solid lipid nanoparticles and their pharmacokinetic behaviors / 中成药

AIM To prepare chrysin solid lipid nanoparticles and to evaluate their pharmacokinetic behaviors.METHODS The particle size,Zeta potential and in vitro release rate of nanoparticles prepared by emulsification uhrasonication-low temperature solidification method were determined.Twelve SD rats were randomly divided into two groups and were intragastrically given suspensions of crude drug and nanoparticles,respectively.HPLC was used for the content determination of chrysin in plasma,after which blood drug concentration-time curves were drawn,and pharmacokinetic parameters were calculated.RESULTS The obtained nanoparticles demonstrated the particle size of (207.15 ±30.59) nm,PDI of (0.224 ±0.067) and Zeta potential of (-34.8 ±5.9) mV,respectively,whose accumulative release rate reached 84.36％ within 36 h.Their Cmax [(9.04 ± 1.52) μg/mL] and AUC0～t,[(33.67 ± 3.47) μg · h/mL] were much higher than those of the crude drug (P ＜ 0.01).CONCLUSION Solid lipid nanoparticles can promote the oral absorption and bioavailability of chrysin,with significant sustained-release characteristics.

Scaled-up production and application of co-processed excipient mannitol-HPMC in traditional Chinese medicine / 中国中药杂志

Spray drying technology was used to produce co-processed excipients mannitol- hydroxypropyl methylcellulose (HPMC), and study the scaled-up production. The consistency of powder and tablet properties before and after scale-up of co-processed excipients was compared, and their applicability in traditional Chinese medicine (TCM) powder's direct compression was tested on five TCM extracts such as gardenia extract and Radix Paeoniae Alba extract. It was shown that after scaled-up production, the key properties of co-processed excipients had little changes (such as compactability, disintegrating time, and lubrication sensitivity) or improvement (such as flowability and yield). As compared to commercially available spray-dried mannitol, co-processed excipients achieved better compactability and higher drug loading for direction compression of TCM powder. In conclusion, the mannitol-HPMC co-processed excipient, with excellent physicomechanical properties, is promising to be explored as a new excipient for direct powder compression.

Development and homeostasis of taste buds in mammals / 华西口腔医学杂志

Taste is mediated by multicellular taste buds distributed throughout the oral and pharyngeal cavities. The taste buds can detect five basic tastes: sour, sweet, bitter, salty and umami, allowing mammals to select nutritious foods and avoid the ingestion of toxic and rotten foods. Once developed, the taste buds undergo continuous renewal throughout the adult life. In the past decade, significant progress has been achived in delineating the cellular and molecular mechanisms governing taste buds development and homeostasis. With this knowledges and in-depth investigations in the future, we can achieve the precise management of taste dysfunctions such as dysgeusia and ageusia.

Identification of HLA-A3 restricted cytotoxic T-lymphocyte epitopes from cancer-testis antigen MAGEC2 / 中国病理生理杂志

AIM:To predict and identify an HLA-A3 supertype-restricted cytotoxic T-lymphocyte(CTL) epitope derived from MAGEC2,which is utility in epitope design for the development of HLA-based vaccines and immuno-therapeutics.METHODS:HLA-A3 epitopes from MAGEC2 protein were predicted by BIMAS, SYFPEITHI and IEDB. The binding affinity of the peptides to HLA-A*03 molecule was evaluated by T2A3 cell binding assay.ELISPOT assay was used to investigate the ability of the peptides inducing specific restricted CTLs to release interferon -γ(IFN-γ).The ability of the peptides to induce T-cell response was investigated by cytotoxicity assay in vitro.RESULTS:The candidate peptides P147,P167, P196, P229 and P251 showed moderate affinity toward HLA-A3 molecule.ELISPOT assay showed that P167,P196 and P251 were able to induce specific CTLs and higher levels of IFN-γwere released.The CTLs induced by P196 and P251 were able to lyse target cells.CONCLUSION:The peptides P196 and P251 have higher binding affinity with HLA-A3 and retain immunogenicity.They are excellent HLA-A3-restricted CTL epitopes from tumor antigen MA-GEC2,which could serve as new candidates towards antitumor peptide vaccines.

Study on preparation and storage stability of ginsenoside Re liposomes / 中草药

Objective To establish and optimize preparation technology of ginsenoside Re liposomes, therefore to improve storage stability. Methods Ginsenoside Re liposomes were prepared by the method of film dispersion-mechanical vibration, which were collected by separating liposome from disclosed free drug by dialysis method. Measure entrapment efficiency by HPLC. Prepare freeze-dried liposome preparations by freezing-drying technology. Taking entrapment efficiency as the main screening index, optimize liposome formulation and freezing-drying technology by orthogonal test design. Results The entrapment efficiency of ginsenoside Re lipidosomes prepared by the method of film dispersion-mechanical vibration is the highest. The best formulation technology is: Mass ratio of drug and phospholipid is 1∶30, mass ratio of phospholipid and cholesterol is 16∶1, ice-water bath ultrasound is 30 min, and double distilled water is hydration solution; The best freezing-drying technology is: Taking sucrose as the freeze-drying protective agent, mass ratio of disaccharide-water is 1∶10, pre-freezing temperature is −20 ℃, and normal saline of 0.9% is reconstitution solution. Conclusion The preparation technology of liposome is stable and practicable. The ginsenoside Re liposome prepared by taking the sucrose as the freeze-drying protective agent has good indexes, which can extend the storage period.

Effects of monosulfuron-ester on metabolic processes of nitrogen-fixing cyanobacteria Anabaena flos-aquae and Anabaena azotica

Abstract Presence of the relatively new sulfonylurea herbicide monosulfuron-ester at 0.03-300 nmol/L affected the growth of two non-target nitrogen-fixing cyanobacteria (Anabaena flos-aquae and Anabaena azotica) and substantially inhibited in vitro Acetolactate synthase activity, with IC50 of 3.3 and 101.3 nmol/L for A. flos-aquae and A. azotica, respectively. Presenting in 30-300 nmol/L, it inhibited protein synthesis of the cyanobacteria with less amino acids produced as its concentration increased. Our findings support the view that monosulfuron-ester toxicity in both nitrogen-fixing cyanobacteria is due to its interference with protein metabolism via inhibition of branch-chain amino acid biosynthesis, and particularly Acetolactate synthase activity.

Effect of glutaryl-CoA dehydrogenase gene silencing and high-concentration lysine on the viability of BRL hepatocytes / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the effect of glutaryl-CoA dehydrogenase (GCDH) gene silencing and accumulation of lysine metabolites on the viability of hepatocytes.</p><p><b>METHODS</b>BRL cells were divided into normal control group, negative control group, and GCDH silencing group. The shRNA lentiviral vector for silencing GCDH gene was constructed, and the BRL hepatocytes in the GCDH silencing group and the negative control group were infected with this lentivirus and negative control virus respectively, and then cultured in a medium containing 5 mmol/L lysine. Immunofluorescence assay was used to measure the infection efficiency of lentivirus. Western blot was used to measure the expression of GCDH protein. MTT assay was used to evaluate cell viability. Hoechest33342 staining was used to measure cell apoptosis. Western blot was used to measure the expression of Caspase-3, an index of cell apoptosis.</p><p><b>RESULTS</b>The lentivirus constructed effectively silenced the GCDH gene in hepatocytes (P<0.01). MTT assay and Hoechest 33342 staining showed no significant differences in cell viability and apoptosis between groups (P>0.05). There was also no significant difference in the expression of Caspase-3 protein between groups (P>0.05).</p><p><b>CONCLUSIONS</b>GCDH gene silencing and accumulation of lysine metabolites may not cause marked hepatocyte injury.</p>

Characteristics of Balance and Surface Electromyography of Trunk Flexor and Extensor for Stroke Patients / 中国康复理论与实践

Objective To study balance function and surface electromyography (sEMG) activity of erector spinae (ES) and rectus ab-dominis(RA)in stroke patients.Methods From April,2015 to December,2016,17 stroke patients(test group)and 13 healthy subjects(con-trol group)were tested with balance platform:sitting with eyes open/close,with the platform swing of 10°,with trunk flexion and extension in maximum.The root mean square(RMS)of sEMG was recorded on both sides of ES and RA synchronously.The swing length and area of center of mass,the center of pressure on X and Y axes were also recorded as sitting with eyes open/close.Results For the test group,RMS of bilateral ES was significantly different as sitting with eyes open/close and with trunk flexion and extension in maximum (t>2.173, P<0.05).Compared with the control group,RMS of ES and RA in unaffected side increased as sitting with eyes open/close(t>2.175,P<0.05), as well as the swing length and area of center of mass(t>2.760,P<0.05).Conclusion The strength imbalance is found in ES in stroke pa-tients,which may associate with the balance function impairment,and need to be improved in rehabilitation.

Efficacy and safety of paroxetine in treatment of post-stroke depression and anxiety / 药物评价研究

Objective To investigate the efficacy and safety of paroxetine in the treatment of post-stroke depression and anxiety.Methods Patients with post-stroke depression and anxiety (312 cases) were randomly divided into observation and control groups.All patients were given routine,symptomatic,supportive treatment and rehabilitation training,psychological intervention,in addition,the observation group was treated with paroxetine hydrochloride.The Hamilton Depression Scale (HAMD) score,anxiety scale (HAMA) score,plasma substance P (SP),neuropeptide Y (NPY),cortisol (Cor) content and side effects scale (TESS) score of both groups before and after treatment were analyzed and compared.Results After treatment,the HAMD and HAMA scores of two groups were significantly decreased,and those of the observation group was significantly lower than the control group (P ＜ 0.05).The clinical curative effect of the observation group was better than that of the control group (P ＜ 0.05).In both groups,the levels of SP and Cor were significantly decreased,NPY was significantly increased,and the improvement of the observation group was higher than that of the control group (P ＜ 0.05).In the observation group,some patients had anorexia,nausea,dizziness,insomnia and other reactions,but they were tolerant,and there was no significant difference in the TESS score between the observation group and control group.Conclusion Paroxetine treatment of post-stroke depression/anxiety can reduce depression and anxiety symptoms,improve neuropeptide and cortisol levels,and have good curative effect and good safety.It is worthy of clinical application and further study.

Endoscopic clipping with histoacryl in management of type 2 gastroesophageal varices (30 cases) / 中国内镜杂志

Objective To summarize clinical experience and explore application value of endoscopic clipping with histoacryl using in management of type 2 gastroesophageal varices. Methods Clinical data of 30 patients with type 2 gastroesophageal varices patients (including acute hemorrhage and primary prevention) from May 2015 to December 2016 were collected. Then evaluate therapeutic effect and safety of endoscopic clipping adjuvant therapy. Results Average glue dosage was (1.46 ± 0.70) ml, average using of clips were (5 ~ 6), and intraoperative needle pulling hemorrhage occurred in 2 cases. 14 patients (46.7%) underwent endoscopic re-examination, 3 patients (10.0%) achieved varicose vein elimination, 11 cases (36.7%) remained residual. Rebleeding occurred in 4 cases (13.3%), and 2 cases died (6.7%), one because of postoperative hematemesis and hemorrhagic shock, the other one died of spontaneous peritonitis and septic shock. For general curative effect, 2 cases (6.7%) were healed, 22 cases (73.3%) were improved, and 6 cases were unhealed (20.0%, 4 cases occurred rebleeding, 2 cases died); 17 cases underwent CT portal venograpy, abnormal embolization was not found in any patients, glue extrusion bleeding occurred in 1 case (3.3%), no patients had severe postoperative complications. Conclusion Endoscopic clipping with histoacryl can be used in the prevention and treatment of type 2 gastroesophageal varices to improve the treatment effect and reduce postoperative bleeding risk, may have good clinical practice value.

Effect of Mac-1 deficiency on the tumor growth of melanoma in mice / 中国比较医学杂志

Objective To explore the influence of Mac-1 deficiency on tumor growth of melanoma. Methods The population of Mac-1 gene knock-out ( Mac-1 -/ -) mice was expanded. B16-F10 cells were subcutaneously injected into the C57BL/6J mice (control group) and Mac-1 -/ -mice (experiment group), respectively. Subsequently,the survival rate, tumor volume and body weight were recorded. The proliferation and infiltration of macrophages were detected by immunohistochemistry. Results The survival rate of Mac-1 -/ - mice was significantly improved compared with the C57BL/6J mice (P ﹤0. 001). The tumor volume and body weight were remarkably decreased in the Mac-1 -/ - mice compared with the control group (P﹤0. 001). Meanwhile, the tumor cell proliferation index was decreased in the Mac-1 -/ - mice compared with the control group (P﹤0. 01). Furthermore, the infiltration of macrophages in the tumor tissues was also decreased in Mac-1 -/ - tumor mice compared with control group. Conclusions Mac-1 gene deletion can significantly suppress melanoma growth.

Effect of Mac-1 deficiency on the tumor growth of melanoma in mice / 中国比较医学杂志

Objective To explore the influence of Mac-1 deficiency on tumor growth of melanoma. Methods The population of Mac-1 gene knock-out ( Mac-1 -/ -) mice was expanded. B16-F10 cells were subcutaneously injected into the C57BL/6J mice (control group) and Mac-1 -/ -mice (experiment group), respectively. Subsequently,the survival rate, tumor volume and body weight were recorded. The proliferation and infiltration of macrophages were detected by immunohistochemistry. Results The survival rate of Mac-1 -/ - mice was significantly improved compared with the C57BL/6J mice (P ﹤0. 001). The tumor volume and body weight were remarkably decreased in the Mac-1 -/ - mice compared with the control group (P﹤0. 001). Meanwhile, the tumor cell proliferation index was decreased in the Mac-1 -/ - mice compared with the control group (P﹤0. 01). Furthermore, the infiltration of macrophages in the tumor tissues was also decreased in Mac-1 -/ - tumor mice compared with control group. Conclusions Mac-1 gene deletion can significantly suppress melanoma growth.

Berberine regulates type 2 diabetes mellitus related with insulin resistance / 中国中药杂志

Insulin resistance (IR) is defined as a series of clinical manifestations for diminished effectiveness of insulin in lowering blood sugar levels caused by decreased sensitivity to insulin of liver, muscle and adipose tissue. IR is the major contributor to the etiology and pathogenesis of type 2 diabetes mellitus (T2DM). Berberine, a traditional Chinese herb extract, has been shown to be safe and effective in lowering blood sugar, alleviating insulin resistance and moderating type 2 diabetes mellitus and its complications. The bioavailability of berberine is extremely low, suggesting that it may play a role in lowering blood sugar and lipid by regulating intestinal flora. Intestinal microbiota may serve as a new potential target for berberine treatment of type 2 diabetes mellitus.