Objective To evaluate the safety and efficacy of a self-developed micro-normothermic machine perfusion (NMP) system (micro-perfusion device) for preserving isolated porcine limbs. Methods Five healthy Landrace pigs were selected, and their left and right forelimbs were randomly divided into the NMP group and static cold storage (SCS) group. The NMP group was perfused with the self-developed micro-perfusion device and polymerized hemoglobin perfusate for 32 hours at normothermia, while the SCS group was preserved at 4 ℃. Hemodynamic parameters such as perfusion pressure and flow were monitored. The pH value, partial pressure of oxygen (PO₂), lactic acid (Lac), creatine kinase (CK) and lactate dehydrogenase (LDH) in the perfusate were measured. Hematoxylin-eosin staining was used to assess the muscle tissue structure, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling was employed to evaluate muscle cell apoptosis, and immunohistochemistry staining was applied to detect the expressions of tumor necrosis factor (TNF)-α and interleukin (IL)-6. A mixed-effects model was used to analyze the effects of time and treatment methods on tissue structure, cell apoptosis and inflammatory factors. Results The device could stably maintain a perfusion pressure of (69±15) mmHg and a flow rate of (117±42) mL/min. The pH value and electrolytes of the perfusate were generally stable, with PO₂ maintained at a high level. Lac was maintained at 5.38(3.81, 6.45) mmol/L, while CK and LDH increased over time. After 32 hours of perfusion in the NMP group, both the myocyte spacing and apoptosis rate were better than those in the SCS group. Mixed-effects model analysis showed that there were statistically significant differences in the effects of NMP treatment and SCS treatment on myocyte spacing and apoptosis rate per unit time (both P < 0.05). There were no statistically significant differences in TNF-α and IL-6 between the two groups, and mixed-effects model analysis showed no statistically significant differences in the effects of NMP treatment and SCS treatment on TNF-α and IL-6 per unit time (both P > 0.05). Conclusions The micro-perfusion device used in this study may achieve 32-hour normothermic preservation in a porcine limb amputation model, maintain basic metabolism and ionic homeostasis, reduce muscle structural damage and cell apoptosis without inducing additional inflammatory responses. This technology is expected to significantly extend the time window for replantation of amputated limbs in disaster rescue and long-distance transportation, providing an important technical basis for clinical translation and subsequent replantation research.

Objective To explore the protective effects of genetically modified porcine erythrocyte suspension as a subnormothermic normoxic mechanical perfusate on hypoxic-ischemic brain injury in cynomolgus monkeys caused by traumatic hemorrhage.Methods Cynomolgus monkeys were randomly divided into positive and negative control groups(a total of 3 monkeys,with 3 left cerebral hemispheres as the positive control group and 3 right cerebral hemispheres as the negative control group)and the subnormothermic perfusion group(n=3).The positive control group was directly sampled 1 hour after circulatory arrest,while the negative control group was placed at subnormothermic conditions for 6 hours after circulatory arrest.The subnormothermic perfusion group underwent 6 hours of subnormothermic normoxic mechanical perfusion of the bilateral common carotid arteries of the cynomolgus monkey hypoxic-ischemic brain injury model using genetically modified porcine erythrocyte suspension 1 hour after circulatory arrest.Before perfusion,cross-matching experiments were conducted between the six genetically modified pig and the cynomolgus monkeys.After the start of perfusion,the levels of routine blood indicators in the perfusate were detected at 0,1,2,3,4,5 and 6 hours.Blood oxygen saturation was recorded,and the levels of Na+,K+,Ca2+,glucose and blood pH in the perfusate were measured,as well as the levels of IgG and IgM in the perfusate.After 6 hours of perfusion,the water content of the brain tissue was measured.Nissl staining was performed on the frontal cortex and hippocampal regions,and immunofluorescence staining was used to detect the expression of glial fibrillary acidic protein(GFAP),ionized calcium-binding adapter molecule 1(Iba1)and neuronal nuclear antigen(NEUN).Results The cross-matching results between the six genetically modified pig and the cynomolgus monkeys were negative.The number of red blood cells in the perfusate decreased significantly at 3 hours of perfusion,and the hemoglobin level showed a downward trend at 1,3,5 and 6 hours.The number of white blood cells and platelets decreased at all time points.The blood oxygen saturation in the subnormothermic perfusion group remained stable at 95%-98%,and the levels of blood oxygen saturation,Na+,Ca2+,glucose and pH were stable,while the K+level first increased and then decreased.There was no significant difference in the levels of IgG and IgM before and after perfusion.The water content of brain tissue at the end of perfusion in the subnormothermic perfusion group was significantly higher than that in the positive control group(P<0.001).Nissl staining results showed that compared with the positive control group,the pyramidal neurons in the prefrontal cortex of the subnormothermic perfusion group maintained better morphological integrity,with no significant increase in enlarged and deformed cells.In the hippocampal CA1 region,there was a slight increase in enlarged and deformed cells,and a few cells with undamaged structures showed reduced cell size.In the hippocampal dentate gyrus,fewer granule neurons had compromised structural integrity,with increased cell edema.NEUN immunofluorescence staining showed that compared with the positive control group,the pyramidal neurons in the prefrontal cortex and hippocampal CA1 region of the subnormothermic perfusion group had better morphological states,with clear axons.The granule cells in the hippocampal dentate gyrus were well preserved,but the nuclei were less well protected.GFAP immunofluorescence staining showed that compared with the positive control group,the subnormothermic perfusion group had sparser protrusions that were more tightly associated with neurons.Iba1 immunofluorescence staining showed that compared with the positive control group,the subnormothermic perfusion group had thicker and fewer protrusions.Conclusions Compared with the positive control group,subnormothermic normoxic mechanical perfusion with genetically modified porcine erythrocyte perfusate increases brain tissue edema in cynomolgus monkeys,but better preserves the morphological integrity of neurons and glial cells.The protective effects may be related to the continuous oxygen and energy supply,maintenance of ion homeostasis and perfusate pH,reduced rejection,and low metabolic state of the whole brain.

Objective To explore the protective effects of genetically modified porcine erythrocyte suspension as a subnormothermic normoxic mechanical perfusate on hypoxic-ischemic brain injury in cynomolgus monkeys caused by traumatic hemorrhage.Methods Cynomolgus monkeys were randomly divided into positive and negative control groups(a total of 3 monkeys,with 3 left cerebral hemispheres as the positive control group and 3 right cerebral hemispheres as the negative control group)and the subnormothermic perfusion group(n=3).The positive control group was directly sampled 1 hour after circulatory arrest,while the negative control group was placed at subnormothermic conditions for 6 hours after circulatory arrest.The subnormothermic perfusion group underwent 6 hours of subnormothermic normoxic mechanical perfusion of the bilateral common carotid arteries of the cynomolgus monkey hypoxic-ischemic brain injury model using genetically modified porcine erythrocyte suspension 1 hour after circulatory arrest.Before perfusion,cross-matching experiments were conducted between the six genetically modified pig and the cynomolgus monkeys.After the start of perfusion,the levels of routine blood indicators in the perfusate were detected at 0,1,2,3,4,5 and 6 hours.Blood oxygen saturation was recorded,and the levels of Na+,K+,Ca2+,glucose and blood pH in the perfusate were measured,as well as the levels of IgG and IgM in the perfusate.After 6 hours of perfusion,the water content of the brain tissue was measured.Nissl staining was performed on the frontal cortex and hippocampal regions,and immunofluorescence staining was used to detect the expression of glial fibrillary acidic protein(GFAP),ionized calcium-binding adapter molecule 1(Iba1)and neuronal nuclear antigen(NEUN).Results The cross-matching results between the six genetically modified pig and the cynomolgus monkeys were negative.The number of red blood cells in the perfusate decreased significantly at 3 hours of perfusion,and the hemoglobin level showed a downward trend at 1,3,5 and 6 hours.The number of white blood cells and platelets decreased at all time points.The blood oxygen saturation in the subnormothermic perfusion group remained stable at 95%-98%,and the levels of blood oxygen saturation,Na+,Ca2+,glucose and pH were stable,while the K+level first increased and then decreased.There was no significant difference in the levels of IgG and IgM before and after perfusion.The water content of brain tissue at the end of perfusion in the subnormothermic perfusion group was significantly higher than that in the positive control group(P<0.001).Nissl staining results showed that compared with the positive control group,the pyramidal neurons in the prefrontal cortex of the subnormothermic perfusion group maintained better morphological integrity,with no significant increase in enlarged and deformed cells.In the hippocampal CA1 region,there was a slight increase in enlarged and deformed cells,and a few cells with undamaged structures showed reduced cell size.In the hippocampal dentate gyrus,fewer granule neurons had compromised structural integrity,with increased cell edema.NEUN immunofluorescence staining showed that compared with the positive control group,the pyramidal neurons in the prefrontal cortex and hippocampal CA1 region of the subnormothermic perfusion group had better morphological states,with clear axons.The granule cells in the hippocampal dentate gyrus were well preserved,but the nuclei were less well protected.GFAP immunofluorescence staining showed that compared with the positive control group,the subnormothermic perfusion group had sparser protrusions that were more tightly associated with neurons.Iba1 immunofluorescence staining showed that compared with the positive control group,the subnormothermic perfusion group had thicker and fewer protrusions.Conclusions Compared with the positive control group,subnormothermic normoxic mechanical perfusion with genetically modified porcine erythrocyte perfusate increases brain tissue edema in cynomolgus monkeys,but better preserves the morphological integrity of neurons and glial cells.The protective effects may be related to the continuous oxygen and energy supply,maintenance of ion homeostasis and perfusate pH,reduced rejection,and low metabolic state of the whole brain.

The outbreak and spread of coronavirus disease 2019 (COVID-19) highlighted the importance and urgency of the research and development of therapeutic drugs. Very early into the COVID-19 pandemic, China has begun developing drugs, with some notable progress. Herein, we summarizes the anti-COVID-19 drugs and promising drug candidates originally developed and researched in China. Furthermore, we discussed the developmental prospects, mechanisms of action, and advantages and disadvantages of the anti-COVID-19 drugs in development, with the aim to contribute to the rational use of drugs in COVID-19 treatment and more effective development of new drugs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the variants. Neutralizing antibody is an effective approach to overcome COVID-19. However, drug resistance induced by rapid virus mutation will likely to challenge neutralizing antibodies. Taking into account current epidemic trends, small molecule drugs have a crucial role in fighting COVID-19 due to their significant advantage of convenient administration and affordable and broad-spectrum. Traditional Chinese medicines, including natural products and traditional Chinese medicine prescriptions, contribute to the treatment of COVID-19 due to their unique mechanism of action. Currently, the research and development of Chinese anti-COVID-19 drugs have led to some promising achievements, thus prompting us to expect even more rapidly available solutions.

Objective To investigate the predictive value of depth of invasion (DOI) of tongue squamous cell carcinoma (TSCC) for cervical lymph node metastasis and prognosis. Methods We retrospectively analyzed the clinical and pathological data of 73 patients with T_1/2 TSCC. ROC curve was used to determine the optimal cut-off value of DOI for predicting cervical lymph node metastasis, and logistic regression analysis was performed to analyze the related factors affecting cervical lymph node metastasis of TSCC. Kaplan-Meier method and Cox regression analysis were used for survival analysis. Results Among 73 patients, 18 patients were with lymph node metastasis and 55 patients were without lymph node metastasis. The median DOI with and without lymph node metastasis were 8.00 and 5.00 mm, respectively (P=0.003). The optimal cut-off value for DOI was 6.15 mm, with AUC 0.75 (95%CI: 64.1%~87.1%, P=0.001), sensitivity 77.8% and specificity 63.6%. DOI and pathological differentiation were independent prognostic factors for cervical lymph node metastasis in multivariate analysis. DOI, nerve invasion and pathological differentiation were independent prognostic factors of survival in Cox regression analysis. Conclusion DOI of TSCC patients has important predictive value for both cervical lymph node metastasis and prognosis. Neck lymph node dissection is recommended for patients with DOI > 6.15 mm to improve survival rate and reduce recurrence rate.

Objective:To investigate the application value of Hisense computer-assisted surgery system (CAS) three-dimensional reconstruction in the precision treatment of pediatric liver tumors.Methods:The retrospective and descriptive study was conducted. The clinicopathological data of 82 children with liver tumors who were admitted to Affiliated Hospital of Qingdao University from January 2013 to September 2021 were collected. There were 39 males and 43 females, aged 13(19)months. Children underwent upper abdominal dynamic enhanced computed tomography (CT) examination, and three-dimensional reconstruction was performed on CT images of arterial, equili-brium and venous phases with Hisense CAS. Surgical feasibility and scheme were evaluated and conducted based on the results of upper abdominal dynamic enhanced CT examination, and then revised according to three-dimensional reconstruction results of Hisense CAS. Observation indicators: (1) comparison of surgical scheme between two-dimensional enhanced CT images and three-dimensional reconstruction results of Hisense CAS; (2) intraoperative and postoperative situations; (3) follow-up. Follow-up was conducted by outpatient examination to detect postoperative compli-cations and residual liver compensation up to November 2021. Measurement data with normal distribution were represented as Mean± SD. Measurement data with skewed distribution were represented as M(IQR) or M(range). Count data were described as absolute numbers or percentages. Results:(1) Comparison of surgical scheme between two-dimensional CT images and three-dimensional reconstruction results of Hisense CAS. ① Based on the two-dimensional CT images, 42 cases of 82 children could undergo one-stage resection and 40 children could not. However, based on the three-dimensional reconstruction results of Hisense CAS, 48 cases of 82 children could undergo one-stage resection and 34 children could not. There were 6 children with one-stage resection feasibility based on the three-dimensional reconstruction results of Hisense CAS rather than the two-dimensional CT images. For the 34 children undergoing chemotherapy firstly, 31 cases had surgical feasibility after chemotherapy based on the two-dimensional CT images and 3 cases could not undergo surgery because of unapparent tumor regression or tumor surrounding impor-tant vessels. However, the 34 children had surgical feasibility after chemotherapy based on the three-dimensional reconstruction results of Hisense CAS. The revision rate of surgical feasibility was 11.0%(9/82) for the 82 children. ② Based on the two-dimensional CT images, 15 cases of 82 children underwent liver left lobectomy, 21 cases underwent liver right lobectomy, 7 cases underwent mesohepatectomy, 13 cases underwent extended left hemihepatectomy, 23 cases underwent extended right hemihepatectomy, 3 cases underwent segmental hepatectomy. However, based on the three-dimensional reconstruction results of Hisense CAS, 20 cases of 82 children underwent liver left lobectomy, 29 cases underwent liver right lobectomy, 7 cases underwent mesohepatectomy, 7 cases underwent extended left hemihepatectomy, 14 cases underwent extended right hemihepatectomy, 5 cases underwent segmental hepatectomy. The revision rate of surgical scheme was 36.6%(30/82) for the 82 children. (2) Intraoperative and postoperative situations. The operation time, volume of intraoperative blood loss, duration of postoperative hospital stay of the 82 children were (182±18)minutes, 20(10)mL, (10.2±1.9)days, respectively. (3) Follow-up. All the 82 children were followed up for 10 (range, 2?18)months. There was no obvious complication occurred to the 82 children after surgery, and the residual liver can satisfy the liver compensation of body. All the children survived well.Conclusion:Three-dimensional reconstruction of Hisense CAS is conducive to judging the surgical feasibility and formulation of accurate surgical plan of children with liver tumors.

OBJECTIVE: To determine the carrier rate of deafness-related genetic variants among 53 873 newborns from Zhengzhou. METHODS: Heel blood samples of the newborns were collected with informed consent from the parents, and 15 loci of 4 genes related to congenital deafness were detected by microarray. RESULTS: In total 2770 newborns were found to carry deafness-related variants, with a carrier rate of 5.142%. 1325 newborns (2.459%) were found to carry heterozygous variants of the GJB2 gene, 1071 (1.988%) were found with SLC26A4 gene variants, 205 were found with GJB3 gene variants (0.381%), and 120 were found with 12S rRNA variants (0.223%). Five newborns have carried homozygous GJB2 variants, two have carried homozygous SLC26A4 variants, five have carried compound heterozygous GJB2 variants, and four have carried compound heterozygous SLC26A4 variants. 33 neonates have carried heterozygous variants of two genes at the same time. CONCLUSION The carrier rate of deafness-related variants in Zhengzhou, in a declining order, is for GJB2, SLC26A4, GJB3 and 12S rRNA. The common variants included GJB2 235delC and SLC26A4 IVS7-2A>G, which are similar to other regions in China. To carry out genetic screening of neonatal deafness can help to identify congenital, delayed and drug-induced deafness, and initiate treatment and follow-up as early as possible.

Objective To explore the prognostic values of telomerase reverse transcriptase promoter (TERTp) mutation and 1p/19q co-deletion in newly-diagnosed O6-methylguanine-DNA methyltransferase (MGMT) promoter un-methylated/isocitrate dehydrogenase (IDH) wild-type glioblastoma multiform (GBM). Methods A total of 82 patients pathologically newly-diagnosed MGMT promoter un-methylated/IDH wild-type GBM, admitted to our hospitals from March 2016 to November 2018, were included in this study. TERTp mutations (TERTp wild-type and TERTp mutation [C228 mutation and C250 mutation]) in GBM specimens were detected by PCR sequencing, 1p/19q co-deletion in GBM specimens was detected by fluorescence in situ hybridization (FISH), and clinical data, adverse reactions and prognoses of patients with different molecular typing were compared. Results There were 33 patients in the TERTp wild type group with mean age of 48 years, and 49 patients in the TERTp mutation group with mean age of 59 years; the difference of age was significant (P<0.05); there were no statistical differences in gender distribution, Karnofsky performance status (KPS) scores, tumor sites and surgical resection degrees between the two groups (P>0.05). There were 8 patients with 1p/19q co-deletion and 74 patients without 1p/19q co-deletion; no significant differences in above clinical parameters were noted between the two groups. There were no statistically significant differences in the incidences of bone marrow suppression, digestive tract response and fatigue, disease progression rate, or survival rate between patients from TERTp wild type group and TERTp mutation group, and between patients with 1p/19q co-deletion and patients without 1p/19q co-deletion (P>0.05). No significant differences in above clinical parameters, disease progression rate, and survival rate were noted between patients with C228 mutation and C250 mutation (P>0.05). Conclusion TERTp typing and 1p/19q co-deletion status do not have prognostic value in newly-diagnosed MGMT un-methylated/IDH wild-type GBM patients; patients with TERTp mutations have older age than wild-type patients; patients with C250 mutation trend to have higher survival rate than those with C228 mutation.

Objective: To study the segment of liver according to the large amount of three-dimensional(3D) reconstructive images of normal human livers and the vascular system, and to recognize the basic functional liver unit based on the anatomic features of the intrahepatic portal veins. Methods: The enhanced CT primitive DICOM files of 1 260 normal human livers from different age groups who treated from October 2013 to February 2017 provided by 16 hospitals were analyzed using the computer-aided surgery system.The 3D liver and liver vascular system were reconstructed, and the digital liver 3D model was established.The vascular morphology, anatomical features, and anatomical distributions of intrahepatic portal veins were statistically analyzed. Results: The digital liver model obtained from the 3D reconstruction of CAS displayed clear intrahepatic portal vein vessels of level four.Perform a digital liver segments study based on the analysis of level four vascular distribution areas.As the less anatomical variation of left hepatic portal vein, the liver was classified into four types of liver segmentation mainly based on right hepatic portal vein.Type A was similar to Couinaud or Cho′s segmentation, containing 8 segments(537 cases, 42.62%). Type B contained 9 segments as there are three ramifications of right-anterior portal vein(464 cases, 36.82%). The main difference for Type C was the variation of right-posterior portal vein which was sector shape(102 cases, 8.10%). Type D contained the cases with special portal vein variations, which needs three-dimensional simulation to design individualized liver resection plan(157 cases, 12.46%). These results showed that there was no significant difference in liver segmental typing between genders(χ²=2.179, P=0.536) and did not reveal any significant difference in liver segmental typing among the different age groups(χ²=0.357, P=0.949). Conclusions The 3D digital liver model can demonstrate the true 3D anatomical structures, and its spatial vascular variations.The observation of anatomic features, distribution areas of intrahepatic portal veins and individualized liver segmentation achieved via digital medical 3D visualization technology is of great value for understand the complexity of liver anatomy and to guide the precise hepatectomy.

Objective: To evaluate the efficacy and safety of the application of dye-tattooing under ultrasound guidance in preoperative localization of neck recurrences from thyroid cancer. Methods: Between October 2014 to September 2016, 25 patients with 34 lesions were enrolled. There were 22 cases of papillary thyroid carcinoma and three cases of medullary thyroid carcinoma, all of which could not be detected by computed tomography. Surgeons located the recurrent lesions using dye-tattooing under ultrasound guidance along with radiologist three days before the operation. Results: All lesions were successfully located (100%), 32 of which were located directly and two of which were located indirectly. Postoperative pathological examination confirmed 25 metastases of papillary thyroid carcinoma, two metastases of medullary thyroid carcinoma, and seven cases of false positives. The accuracy of ultrasound diagnosis was 79.4%. After 15 months of follow-up, neither tumor residual nor recurrences was detected according to imaging tests. Conclusions Dye-tattooing under ultrasound guidance represents a reliable and safe method for localization of neck recurrences from thyroid cancer. The cooperation between experienced surgeons and radiologists will be crucial to successful location.