Patients with severe trauma require an extremely timely treatment and transfusion plays an irreplaceable role in the emergency treatment of such patients. An increasing number of evidence-based medicinal evidences and clinical practices suggest that patients with severe traumatic bleeding benefit from early transfusion of low-titer group O whole blood or hemostatic resuscitation with red blood cells, plasma and platelet of a balanced ratio. However, the current domestic mode of blood supply cannot fully meet the requirements of timely and effective blood transfusion for emergency treatment of patients with severe trauma in clinical practice. In order to solve the key problems in blood supply and blood transfusion strategies for emergency treatment of severe trauma, Branch of Clinical Transfusion Medicine of Chinese Medical Association, Group for Trauma Emergency Care and Multiple Injuries of Trauma Branch of Chinese Medical Association, Young Scholar Group of Disaster Medicine Branch of Chinese Medical Association organized domestic experts of blood transfusion medicine and trauma treatment to jointly formulate Chinese expert consensus on blood support mode and blood transfusion strategies for emergency treatment of severe trauma patients ( version 2024). Based on the evidence-based medical evidence and Delphi method of expert consultation and voting, 10 recommendations were put forward from two aspects of blood support mode and transfusion strategies, aiming to provide a reference for transfusion resuscitation in the emergency treatment of severe trauma and further improve the success rate of treatment of patients with severe trauma.

Objective:The mouse kidney transplantation model presents challenges in terms of surgical difficulty and low success rate,making it difficult to master.This study aims to provide a crucial model for transplantation immunology research by modifying and developing novel techniques for mouse kidney transplantation. Methods:A total of 57 pairs of mice were used to establish and compare the modified and innovative surgical techniques for mouse kidney transplantation.Three different surgical models were established,including the abdominal suture technique for orthotopic kidney transplantation,the abdominal cuff technique for orthotopic kidney transplantation,and the cervical cuff technique for ectopic kidney transplantation.BALB/c or C57BL/6 male mice,aged 8 to 12 weeks and weighed 20 to 25 g with specified pathogen free-grade were served as the donor mice or the recipient mice.The surgical technique characteristics,key surgical times,complications,and pathological examination in the early postoperative period were summarized and compared. Results:Three different surgical models of mouse kidney transplantation were successfully established.The comparison of warm ischemic time for the 3 groups of mice showed no statistical significance(P=0.510 4).The abdominal suture group had the shortest total operation time of the donor compared with the abdominal cuff group and the cervical cuff group[(18.3±3.6)min vs(26.2±4.7)min and(22.8±2.5)min;both P<0.000 1].There was a significant difference in cold ischemia time among the 3 groups(all P<0.000 1),with(60.8±4.1)min in the cervical cuff group,(43.3±5.0)min in the abdominal suture group,and(88.8±6.7)min in the abdominal cuff group.Due to different anastomosis methods,the cervical cuff group had the shortest time[(17.6±2.7)min],whereas the abdominal cuff group had the longest time[(38.8±5.4)min].The total operation time for the recipients showed significant differences(P<0.000 1),with the abdominal suture group having the shortest time[(44.0±6.9)min],followed by the cervical cuff group[(64.1±5.2)min],and the abdominal cuff group[(80.0±6.0)min]being the longest.In the 32 mice of the abdominal suture group,there were 6 with intraoperative bleeding,including 1 arterial intimal injury bleeding and 5 with bleeding after vessel opening.Six mice had ureteral complications,including ureteral bladder anastomotic stenosis,necrosis,and renal pelvis dilation.Two mice had postoperative abdominal infections.In the abdominal cuff group,there was no intraoperative bleeding,but 6 mice showed mild arterial stenosis and 5 showed venous stenosis,4 arterial injury,4 arterial thrombosis,and 2 ureteral complications.No postoperative infections occurred in the mice.In the cervical cuff group,no intraoperative bleeding,arterial intimal injury,arterial/venous stenosis,or thrombosis were found in 13 mice.Five mice had ureteral complications,including ureteral necrosis and infection,which were the main complications in the cervical cuff group.The renal function in mice of the 3 groups remained stable 7 days after surgery.Hematoxylin and eosin staining and periodic acid-Schiff staining showed no significant differences in terms of acute rejection among the 3 surgical methods(all P>0.05). Conclusion:All 3 surgical methods are able to successfully establish mouse kidney transplantation models,with no significant differences observed in the short-term graft survival and acute rejection.The modified abdominal suture technique and abdominal cuff technique have their respective advantages in research applications.The novel cervical cuff technique for ectopic kidney transplantation model is relatively simple to be prepared and causes less trauma to the mice,providing more options for studies involving xenotransplantation,secondary transplantation,and local lymphatic drainage.However,the difficulty in harvesting the donor kidney and the high incidence of ureteral infections need further validation in long-term survival.This study holds important reference value for choosing the type of mouse kidney transplantation model for different research needs.

Background::Homoharringtonine (HHT) is an effective anti-inflammatory, anti-viral, and anti-tumor protein synthesis inhibitor that has been applied clinically. Here, we explored the therapeutic effects of HHT in a mouse heart transplant model.Methods::Healthy C57BL/6 mice were used to observe the toxicity of HHT in the liver, kidney, and hematology. A mouse heart transplantation model was constructed, and the potential mechanism of HHT prolonging allograft survival was evaluated using Kaplan–Meier analysis, immunostaining, and bulk RNA sequencing analysis. The HHT-T cell crosstalk was modeled ex vivo to further verify the molecular mechanism of HHT-induced regulatory T cells (Tregs) differentiation. Results::HHT inhibited the activation and proliferation of T cells and promoted their apoptosis ex vivo. Treatment of 0.5 mg/kg HHT for 10 days significantly prolonged the mean graft survival time of the allografts from 7 days to 48 days ( P <0.001) without non-immune toxicity. The allografts had long-term survival after continuous HHT treatment for 28 days. HHT significantly reduced lymphocyte infiltration in the graft, and interferon-γ-secreting CD4 + and CD8 + T cells in the spleen ( P <0.01). HHT significantly increased the number of peripheral Tregs (about 20%, P <0.001) and serum interleukin (IL)-10 levels. HHT downregulated the expression of T cell receptor (TCR) signaling pathway-related genes ( CD4, H2-Eb1, TRAT1, and CD74) and upregulated the expression of IL-10 and transforming growth factor (TGF) -β pathway-related genes and Treg signature genes ( CTLA4, Foxp3, CD74, and ICOS). HHT increased CD4 + Foxp3 + cells and Foxp3 expression ex vivo, and it enhanced the inhibitory function of inducible Tregs. Conclusions::HHT promotes Treg cell differentiation and enhances Treg suppressive function by attenuating the TCR signaling pathway and upregulating the expression of Treg signature genes and IL-10 levels, thereby promoting mouse heart allograft acceptance. These findings may have therapeutic implications for organ transplant recipients, particularly those with viral infections and malignancies, which require a more suitable anti-rejection medication.

OBJECTIVE: To explore the genetic basis for 4 patients with globozoospermia. METHODS: Semen and blood samples were collected from the patients for the determination of sperm concentration, viability, survival rate, morphology and acrosome antigen CD46. Meanwhile, DNA was extracted for whole exome sequencing (WES), and candidate variants were validated by Sanger sequencing. RESULTS: All of the four patients were found to harbor variants of the DPY19L2 gene. Patients 1 ~ 3 had homozygous deletions of the DPY19L2 gene. Sanger sequencing confirmed that the DPY19L2 gene in patient 3 was disrupted at a recombination breakpoint area BP2, resulting in nonallelic homologous recombination and complete deletion of the DPY19L2 gene. Patients 2 and 3 respectively harbored novel homozygous deletions of exons 2 ~ 22 and exons 14 ~ 15. Patient 4 harbored heterozygous deletion of the DPY19L2 gene, in addition with a rare homozygous deletion of the 3' UTR region. CONCLUSION DPY19L2 gene variants probably underlay the globozoospermia in the four patients, which has fit an autosomal recessive pattern of inheritance and the characteristics of genomic diseases.
Male ; Humans ; Teratozoospermia/genetics* ; Homozygote ; Semen ; Sequence Deletion ; 3' Untranslated Regions ; Membrane Proteins

Objective:To explore the clinical efficacy of adult donor dual kidney transplantation.Methods:Retrospective analysis of case data of 13 adult donor kidney dual kidney transplantation (DKT) performed in the The Second Xiangya Hospital of Central South University from September 2016 to December 2020. For 13 donors, the average age and BMI were (53.5±12.4)years and (24.3±2.8) kg/m 2, respectively. Their mean Serum creatinine (SCr) at admission and before procurement was (132.9±54.1)and (228.7±112.4)μmol/L, respectively. 3 of them had diabetes mellitus history, and 8 had hypertension history. 11 met the United Network for Organ Sharing (UNOS) DKT criteria and 6 met Remuzzi score DKT criteria. For 13 recipients, the average age and BMI were (39.3±8.9)years and (20.2±2.4)kg/m 2, respectively. All of them received ABO blood type-matched kidney transplants. 2 of them had their grafts transplanted in the bilateral iliac. In 12 cases, the grafts filled rapidly and urinated immediately when opening blood flow. In 1 case, the grafts were dark in color and vascular showed weak pulsation after opening blood flow. The time to recovery of perioperative graft function (from the day of surgery to the natural reduction of SCr to the normal range 44-133μmol/L), the occurrence of delayed graft function (DGF), acute rejection (AR), ureteral and surgical incision complications, as well as the recipients’ final follow-up SCr, eGFR, urinary protein, and grafts outcome were observed. Risk factors affecting outcomes were assessed by univariate logistic regression analysis. Results:The SCr dropped to the normal range at discharge in 10 recipients, and the average recovery time was (13.8±13.0) days. In other 3 cases SCr at discharge were 300.0, 149.0, 152.5μmol/L. 4 cases had DGF, 4 had AR, 1 experienced urinary fistula, and 1 experienced incisional dehiscence, which were treated with anti-rejection, J-tube implantation, continuous catheterization to maintain bladder void, secondary suturing, respectively. The follow-up time ranged from 4 to 54 months, with a median of 28(15.5, 31.0) months. At the final follow-up time, 10 cases had good graft function, 2 suffered impaired kidney function, and 1 experienced graft failure. The average SCr and eGFR except for graft failure patient were (144.2±101.3)μmol/L and (52.9±21.2)ml/min, respectively. 4 had positive urine protein. Univariate logistic regression analysis showed that donor age, BMI, history of diabetes mellitus and hypertension, and SCr were not significantly correlated with recipients’ DGF and graft impairment ( P>0.05), and due to the small sample size, multifactorial logistic regression analysis was not performed. Conclusion:The short to medium-term effects of adult donor DKT coule be safe and feasible.

Objective:To explore clinical value of nucleic acid detection for hepatitis B virus (HBV) screening in hospitalized patients.Methods:This cross-sectional study collected and analyzed plasma samples from patients admitted to 10 domestic medical institutions from July 2021 to December 2021. Serological immunoassay and nucleic acid screening were used to simultaneously detect hepatitis B markers such as hepatitis B surface antigen (HBsAg), hepatitis B surface antibody (HBsAb), hepatitis B e Antigen (HBeAg), hepatitis B e antibody (HBeAb), hepatitis B core antibody (HBcAb),and HBV DNA. Statistical analysis was performed on the serology, nucleic acid test results and clinical information of the patients.Results:Of the 8 655 collected samples, HBsAg was positive in 216 (2.50%) samples,HBV DNA was positive in 238 (2.75%) samples ( P>0.05); 210 (2.43%) samples were positive for both HBsAg and HBV DNA, 28 (0.32%) were HBsAg negative and HBV DNA positive, 6 cases (0.07%) were HBsAg positive and HBV DNA negative. Conclusion:These results indicate that the HBV DNA testing is equally effective as hepatitis B virus serological detection for hepatitis B virus screening in hospitalized patients.

Objective:This multi-centre study was conducted to assess the efficacy of various preoperative/pre-transfusion screening methods for blood transmitted disease.Methods:From July 2021 to December 2021, plasma samples of patients admitted to 10 hospitals were collected for screening preoperative/pre-transfusion blood transmitted disease. Nucleic acid detection technology was used to detect hepatitis B virus (HBV) DNA, hepatitis C virus (HCV) RNA and human immunodeficiency virus (HIV)(1+2) RNA, and the results were compared with the immuno-serological methods. χ 2 test and Kappa test were used to analyze the efficacy of these two methods. Results:A total of 8 655 valid specimens were collected from 10 hospitals. There was a statistically significant difference in the positive detection rate of HCV between the two methods ( P<0.001). There was no significant difference in the positive detection rate of HBV and HIV assessed by the two methods ( P>0.05), but the number of positive cases detected by HBV DNA and HIV RNA (218 and 4 cases) was significantly higher than the corresponding serological results (216 and 2 cases). At the same time, there were HBV, HCV and HIV immuno-serological omissions by the immuno-serological methods, among which 28 cases were HBsAg negative and HBV DNA positive, 2 cases were HCV antibody negative and HCV RNA positive, and 2 cases were HIV antigen/antibody negative and HIV RNA positive. In addition, in the 66 samples with inconsistent results from the two detection methods, 83.3% (55/66), 68.2% (45/66), 63.6% (42/66) and 62.1% (41/66) of patients aged was>45 years, tumor, surgery and male, respectively. Conclusions:Compared with immuno-serological tests, nucleic acid tests have the advantage in terms of sensitivity on detecting HBV, HCV and HIV infection and could reduce missed detection. The risk of transmission can be reduced by adding HBV, HCV, and HIV nucleic acid tests to preoperative/pre-transfusion immuno-serological tests screening for patients over 45 years of age and tumor patients.

Objective:To compare the cost-effectiveness of hospitalized Chinese patients undergoing nucleic acid screening strategies for hepatitis B and hepatitis C, immunological screening strategy, and no screening strategy under different willingness to pay (WTP). The results might aid to decision-making for the optimal strategy.Methods:In this study, nucleic acid screening, immunological screening and no screening were used as screening strategies, and China′s GDP in 2021 (80 976 yuan) was used as the threshold of WTP to construct a Markov model. After introducing parameters related to the diagnosis and treatment of hepatitis B and C in inpatients, a cohort population of 100 000 inpatients was simulated by TreeAge Pro 2021 software, the total cost, total health effects, incremental cost-effectiveness ratio and average cost-effectiveness ratio of different screening strategies were calculated, and cost-effectiveness analysis was conducted. Univariate and probabilistic sensitivity analysis were used to assess the impact of parameter uncertainty on the final results.Results:Compared with the non-screening strategy, the incremental total cost of the hepatitis B immunological screening strategy for cohort patients was 11 049 536 yuan, and the incremental cost-effectiveness ratio was 24 762 yuan/quality-adjusted life years (QALY), while the total incremental cost of nucleic acid screening was 19 208 059 yuan, and the incremental cost-effectiveness ratio was 29 873 yuan/QALY; the incremental cost-effectiveness ratio of nucleic acid screening and immunological screening was 45 834 yuan/QALY. Compared with the non-screening strategy, the incremental cost-effectiveness ratio of hepatitis C immunological screening strategy was 5 731 yuan/QALY, the incremental cost-effectiveness ratio of nucleic acid screening strategy was 8 722 yuan/QALY, the incremental cost-effectiveness ratio of nucleic acid screening and immunological screening was 45 591 yuan/QALY. The results of probabilistic sensitivity analysis showed that when the cost of nucleic acid testing exceeded 214.53 yuan, it was not cost-effective to perform hepatitis B nucleic acid screening under the WTP as 1 fold GDP. When the cost of nucleic acid testing exceeded 132.18 yuan, it was not cost-effective to conduct hepatitis C screening under the WTP as 1 fold GDP.Conclusions:Nucleic acid screening strategy can achieve more cost-effectiveness and is worthy of vigorous promotion. Compared with no screening, both the nucleic acid and immunological screening strategies are cost-effective, and hepatitis nucleic acid screening is the optimal strategy for hospitalized patients.

Objective: Immunoglobulin A nephropathy (IgAN) is one of the most common types of kidney disease, and kidney transplantation is the most effective treatment for end-stage renal disease. This study aims to analyze the clinical curative effect of renal transplantation for adults with IgAN and to discuss the efficacy and safety of kidney transplantation for IgAN at the perioperative period and medium- and long-term follow-up. Methods: This retrospective study included the clinical and follow-up data of 81 adult patients with IgAN who underwent kidney transplantation at the Second Xiangya Hospital, Central South University from January 2018 to January 2022. Of the 81 patients whose age at (34.1±9.9) years old, 47 (58.0％) were male. The body mass index was (20.8±3.2) kg/m2, and the human leukocyte antigen (HLA) mismatch number was 3.5±1.2. The estimated glomerular filtration rate (eGFR) and daily 24-hour urine output for the recipients on the 1st, 5th, and 7th day after kidney transplantation and when they were discharged were analyzed. The recovery of the transplanted kidney and occurrence of complications were comprehensively evaluated. The eGFR, urinary protein, and occult blood were evaluated at the 6th, 12th, 24th, 36th, and 48th month and at the last follow-up. Results: The follow-up time was (25.7±15.8) months. No primary non-function occurred in any patient during the perioperative period time. Fifty-one (63.0％) patients had immediate graft function recovery, and 16 (19.8％) patients had slow graft function recovery. Delayed recovery of graft function was observed in 14 (17.3％) patients. A total of 19 perioperative complications occurred, including 9 patients with acute rejection, 5 patients with urinary fistula, 1 thrombosis in both lower limbs, and 4 lymphatic fistula. The eGFR at 6th, 12th, 24th, 36th, and 48th month of follow-up were (65.3±22.9), (67.6±23.0), (64.3±21.8), (65.9± 24.7), and (68.7±31.2) mL/(min·1.73 m2), respectively. The eGFR remained high during the medium- and long-term follow-ups. At the longest follow-up of 56 months, eGFR fluctuation was still mild, and the positive rate of urine protein and occult blood was low. IgAN recurred in 4 transplanted kidneys, accounting for 4.94％ of the total patients, without severe renal insufficiency. Three patients had kidney dysfunction due to severe pneumonia, rejection, and stone in the transplanted kidney. The overall survival rate of the transplanted kidney was higher than 95％, and the survival rate of all patients was 100％ till Januray 2022. Conclusion: Renal transplantation for adults with IgAN had a remarkable short-term effect. The recipients can be beneficial significantly to favorable midium- and long-term outcomes. IgAN recurrence is infrequent and rarely causes severe renal function damage.

Objective:To investigate the correlation between the prognosis of patients infected with BK virus after renal transplantation and their peripheral blood related indexes.Methods:131 patients from the Renal Transplantation Department of the Second Xiangya Hospital of Central South University who underwent renal transplantation and firstly infected with BK virus after the surgery during the period from August 2018 to August 2021 were retrospectively analyzed. 93 males (71.0%) and 38 females (29.0%). The average age was (37.5±11.3) years old. 109 cases underwent cadaveric kidney transplant (83.2%) and 22 cases underwent relatives kidney transplant (16.8%). The onset time of the first infection with BK virus after renal transplantation was (188.7±16.6) days, and the serum creatinine was (127.5±39.5) μmol/L. 25 patients (19.1%)infected with BK virus were positive in blood and urine at the same time, and 106 patients (80.9%)infected with BK virus were positive only in urine. Among 131 patients infected with BK virus, 70 patients were treated by lowering the blood concentration of tacrolimus to enhance immunity, 12 patients were treated by switching tacrolimus to cyclosporine, and 49 patients had incomplete follow-up data. The DNA load of BK virus in 25 patients [5.6(2.4, 12.3)×10 3copies/ml] positive in blood, white blood cell count(WBC)(5.8±2.0)×10 9/L, hemoglobin(Hb)(122.0±22.4)g/L, platelet count(PLT)(187.1±63.1)×10 9/L, neutrophil count(NEUT)(3.9±1.7)×10 9/L, lymphocyte count(LYM)(1.5±0.8)×10 9/L, monocyte count(MONO)(0.4±0.2)×10 9/L, neutrophil to lymphocyte ratio(NLR)2.2(1.7, 3.5), derived neutrophil to lymphocyte ratio(dNLR)1.7(1.3, 2.6), platelet to lymphocyte ratio(PLR)121.3(86.3, 227.3), monocyte to lymphocyte ratio(MLR)0.2(0.1, 0.4) and lymphocyte to monocyte ratio(LMR)4.7±2.6. The DNA load of BK virus in 106 patients [20.4(0.4, 2 570.0)×10 5copies/ml] positive in urine, WBC 6.6(4.8, 9.1)×10 9/L, Hb(129.0±24.5)g/L, PLT 188.0(147.3, 226.5)×10 9/L, NEUT 4.6(3.0, 6.6)×10 9/L, LYM(1.7±0.8)×10 9/L, MONO 0.4(0.3, 0.5)×10 9/L, NLR 2.8(1.9, 3.9), dNLR 2.1(1.5, 3.0), PLR 120.5(87.0, 163.2), MLR 0.2(0.1, 0.4), LMR 4.5(2.8, 6.7). 70 patients infected with BK virus treated by lowering the blood concentration of tacrolimus were divided into BK virus rise group and BK virus decline group according to the change of BK virus DNA load in blood and urine before and after treatment (the grouping principle of this study gives priority to the change of BK virus DNA load in blood, followed by the change of BK virus DNA load in urine). The WBC, Hb, PLT, NEUT, LYM, MONO, NLR, dNLR, PLR, MLR, LMR, tacrolimus blood concentration and change difference, blood creatinine and change difference were analysed between two groups. Results:The BK virus DNA load in 25 patients positive in blood was correlated with NLR and dNLR ( r=0.5062, P=0.0098; r=0.5738, P=0.0027), and there was no correlation between the BK virus DNA load in blood with the WBC ( r=-0.0185, P=0.9302), Hb ( r=0.0912, P=0.6646), PLT ( r=-0.3931, P=0.0519), NEUT ( r=0.2438, P=0.2401), LYM ( r=-0.3035, P=0.1402), MONO ( r=-0.3279, P=0.1096), PLR( r=0.1054, P=0.6161), MLR( r=0.0738, P=0.7257), LMR( r=-0.0738, P=0.7257). There was no correlation between the BK virus DNA load in 106 patients positive in urine and WBC( r=0.0222, P=0.8209), Hb( r=-0.0323, P=0.7423), PLT( r=0.0847, P=0.3881), NEUT( r=0.0417, P=0.6713), LYM( r=0.0010, P=0.9916), MONO( r=0.0224, P=0.8196, NLR( r=0.0170, P=0.8623), dNLR ( r=-0.0013, P=0.9892), PLR( r=0.0387, P=0.6934), MLR( r=-0.0070, P=0.9433)and LMR( r=0.0070, P=0.9433). As for 70 patients infected with BK virus, there were 37 patients in the BK virus rise group and 33 patients in the BK virus decline group. In the two groups, age [(38.4±12.0)years old and(39.0±9.0)years old], gender [male /female: (23/14) cases and(27/6)cases], blood type [A+ /B+ /AB+ : (22/13/20)cases and (26/6/1)cases], donation type [relatives donnation/cadaveric donation: (29/8)cases and (27/60)cases], blood creatinine(after treatment)[123.0(98.4, 140.5)μmol/L and 132.0(107.1, 162.4)μmol/L] and change difference before and after treatment [0(-15.7, 10.5)μmol/L and -2.0(-9.1, 15.0)μmol/L], tacrolimus blood concentration (after treatment)[(6.7±2.0)ng/ml and(6.5±1.5)ng /ml] and tacrolimus concentration change difference [-1.4(-3.8, 0.6)ng/ml and -1.2(-2.2, 1.3)ng/ml] had no significant difference( P<0.05). The MONO of the two groups was statistically different [0.3(0.2, 0.5)×10 9/L and 0.4(0.3, 0.6)×10 9/L, P=0.033], and there was no difference between the two groups in WBC[6.6(4.1, 8.8)×10 9/L and 6.8(5.4, 8.9)×10 9/L], Hb[(133.2±25.3)g/L and(131.6±20.6)g/L], PLT[185.0(151.0, 231.5)×10 9/L and 196.0(149.0, 234.0)×10 9/L], NEUT[4.3(2.4, 6.4)× 10 9/L and 4.2(3.1, 5.5)×10 9/L], LYM[1.7(1.1, 2.2)×10 9/L and 1.8(1.1, 2.3)×10 9/L], NLR[2.5(1.9, 3.8)and 2.4(1.9, 3.7)], dNLR [2.0(1.5, 2.8)and 1.9(1.4, 2.5)], PLR [114.9(85.1, 159.4)and 111.3(77.1, 159.6)], LMR(4.6±2.6 and 5.2±2.4), MLR[0.2(0.2, 0.4)and 0.2(0.2, 0.4)]( P<0.05). Conclusions:There is a positive correlation between the blood BK virus DNA load and NLR, dNLR in renal transplant recipients infected with BK virus. The rise of MONO correlates with good prognosis of BK virus.