First report of apple root rot caused by Phytopythium vexans in China.

Apple production is of great economic importance in the fruit industry of China, where Yunnan Province is considered as a major producing area. A survey was conducted to identify apple trees that were problematic from March to November 2020 in Yunnan Province. Symptoms included smaller yellowing leaves, fewer sprouts per branch, browning and necrosis of the roots and lower parts of the stem bark, and wilting. 20% to 45% of apple trees were found infected and randomly scattered in the surveyed orchards. A total of 110 soil samples were collected from the root area of symptomatic apple trees in Tuanjie Town of Kunming City, Zhaotong City, and Malong District of Qujing City in Yunnan Province. Two grams of each soil sample was suspended in 400 ml of sterile water for three days and each soil extract was baited with two apple leaves (Red Fuji's). Following the baiting, those leaves were cut into 10 pieces (5mm×5mm), surface-sterilized with 70% ethanol for 30 seconds, rinsed three times with sterile water, and then air-dried. Each leaf piece was placed in a Petri dish with the oatmeal agar medium containing PCNB 20 mg/ml, rifampicin 20 mg/ml, and then incubated at 25℃ in the dark for 3 days. A mycelial agar plug was picked from the edge of the colonies and transferred to a fresh Potato Dexrose Agar (PDA) plate. Seventy colonies with similar growing characteristics were isolated from the 110 soil samples. Three isolates were retained for further analysis and named XLD8-1, SD1, and YF2. After being cultivated on PDA plates and incubated at 25℃ in the dark for 4 days, their colonies were rose petal-type and white with dense aerial hyphae (Fig 1, A). In ten days of incubation, oogonium measuring 24.55 ± 1.9µm × 20.27 ± 2.3µm and sporangia measuring 21.65 ± 1.3µm × 19.35 ± 1µm were observed (Fig 1, C, D). The total DNA of the isolates was extracted and amplified using three pairs of primers, ITS1/ITS4 (White et al. 1990), LROR/LR7 (LSU) (Vilgalys R, et al. 1990), and FM58/FM66 (COXⅡ) (Martin F N. 2000). The sequences were uploaded to GenBank (Accession No. OL960234, OK037658, OK052604 for ITS, OL960388, OM838413, OM838314 for LSU, and OM962847, OM962848, OM962849 for COXⅡ). ITS sequences of the three isolates (XLD8-1, SD1, YF2) showed 99.87%,99.87%, 99.87% similar to Pp. vexans (Accession No. AB468784, AB468784, and AM701801). LSU sequences of the three isolates showed 99.92%, 99.72%, 100% similar to Pp. vexans (Accession No. EF426541, MT729990, and EF426541). COXⅡ sequences of the three isolates showed 100%, 99.81%, 99.81% similar to Pp. vexans (Accession No. GU133560). Based on the sequence similarity and morphology, the isolates were identified as Phytopythium vexans. Koch's postulates were conducted by wounding the bases of 3 apple seedlings (1-year-old Red Fuji's) with a cork borer. A plug of mycelium of the isolate XLD8-1 grown on PDA plates was placed on each wound (Fig 1, B). Controls were set up to use sterile agar plugs as an inoculum. Seedlings have incubated an incubator at 23-26°C under the alternating light and dark intervals, 12-hours of each. In 15 days, after were inoculated with XLD8-1 the roots and lower part of the stem bark of those seedlings became brownish and necrotic, and their epidermis was easily sloughed off (Fig 1, E-G). The pathogen isolated from the necrotic root tissues were identical to the isolate XLD8-1. Symptoms of apple growth decline caused by Pp. vexans were reported in Morocco (Jabiri Salma, et al. 2021). This experiment verified that Pp. vexans causes root rot of apple. In China, Fusarium sp. is usually considered the main pathogen causing apple root rot. However, the discovery of large numbers of apple trees that were infected by Pp. vexans in Yunnan Province and the confirmation of pathogenicity of Pp. vexans on apple seedlings have demonstrated for the first time that Pp. vexans could cause apple root rot as Fusarium spp does and become an incoming threat to the apple industry, which lays the foundation for study on the disease epidemiology and integrated management of apple root rot in China. References: Jabiri Salma, et al. 2021. Microorganisms, doi:10.3390/MICROORGANISMS9091916. Martin, F. N. 2000. Mycologia, 92(4), 711-727. Vilgalys R., et al. 1990. Journal of Bacteriology, 172:4238-4246 White, T. J., et al. 1990. PCR Protocols: a guide to methods and applications, 18: 315.

Metformin inhibits endothelial progenitor cell migration by decreasing matrix metalloproteinases, MMP-2 and MMP-9, via the AMPK/mTOR/autophagy pathway.

The aim of the present study was to investigate the effect of metformin on endothelial progenitor cell (EPC) migration and to explore the possible mechanisms. EPCs were treated with metformin, and the migration of EPCs was evaluated by wound healing and Matrigel invasion assays. We also examined the expression levels of of MMP-2 and MMP-9 in EPCs with or without metformin treatment via RT-PCR and western blot analysis, and activities of MMP-2 and MMP-9 in EPCs under different conditions was examined by zymography. Moreover, we also assessed the AMPK/mTOR/autophagy pathway to explore the possible mechanisms. Metformin treatment significantly downregulated matrix metalloproteinase-2 (MMP-2) and MMP-9 expression, and subsequently decreased the migration of EPCs. Increased levels of phosphorylated (p)-AMPK and LC3II expression, as well as decreased levels of p-mTOR and p62 contributed to this phenomenon. The AMPK inhibitor compound C reversed the effect exerted by metformin. In conclusion, our results showed that metformin inhibited the migration of EPCs by decreasing MMP-2 and MMP-9. The AMPK/mTOR/autophagy pathway was demonstrated to be involved in the regulatory mechanisms.

High expression of CD39 in gastric cancer reduces patient outcome following radical resection.

Ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1), also known as cluster of differentiation (CD)39, is the rate-limiting enzyme in the generation of immunosuppressive adenosine and is important in tumor progression. The present study evaluated the expression of CD39+ and CD39+ forkhead box P3 (FoxP3)+ regulatory T (Treg) cells in gastric cancer (GC), and determined their prognostic roles in patients with GC following radical resection. It was observed that CD39 was expressed at significantly higher rates in tumor tissues as compared with paired peritumoral tissues. Overexpression of tumor CD39 was correlated with overall survival (OS). Furthermore, CD39 expression in GC tissues exhibited a prognostic role in OS. The CD39+ FoxP3+/FoxP3+ ratio in tumor tissues was higher than that in paired peritumoral tissues, and CD39+ FoxP3+ Treg cells were a better prognostic indicator than FoxP3+ Treg cells for OS. Collectively, our study indicates that overexpression of CD39 in GC is a predictor of poor outcome for GC patients following radical resection. CD39+ FoxP3+ Treg cells are a potential target for cancer immunotherapy.

The Value of Performing Early Non-enhanced CT in Developing Strategies for Treating Acute Gallstone Pancreatitis.

BACKGROUND: The purpose of this study is to assess the value of early abdominal non-enhanced computed tomography (NECT) in developing strategies for treating acute gallstone pancreatitis (AGP). METHODS: AGP patients underwent NECT within 48 h after symptom onset to determine the presence of peripancreatic fluid collection, gallstones, and common bile duct stones. Patients with mild AGP who had neither organ failure by clinical data nor peripancreatic fluid collection by NECT (classified as grade A, B, or C based on the Balthazar CT grading system) were randomized to undergo an early laparoscopic cholecystomy (ELC; LC performed within 7 days after a pancreatitis attack, without waiting for symptom resolution) or late laparoscopic cholecystomy (LLC; LC performed ≥ 7 days following an attack, with the patient being completely free of AGP symptoms). RESULTS: The study enrolled 102 patients with mild AGP defined by clinical data and NECT. NECT was 89.2 % and 87.8 % accurate in detecting gallbladder stones and CBD stones, respectively. Totals of 49 and 53 patients were assigned to an ELC and LLC group, respectively. All patients in both groups were cured, no LC-related complications occurred, and no case of AGP increased in severity following LC. The mean lengths of hospital stay and LC operation time were significantly shorter in the ELC group than the LLC group (P < 0.05). CONCLUSIONS: NECT can accurately detect peripancreatic fluid collection and biliary obstructions; thus, early abdominal NECT is valuable when developing strategies for treating AGP. Patients with mild AGP without organ failure or peripancreatic fluid collection can safely undergo ELC without waiting for complete resolution of their pancreatitis.

Overexpression of CD39 and high tumoral CD39⁺/CD8⁺ ratio are associated with adverse prognosis in resectable gastric cancer.

CD39/ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1) is a cell surface-located, rate-limiting enzyme in the generation of adenosine, and plays a crucial role in tumor development. We examined co-expression of CD39 and CD8in gastric cancer (GC) and showed that the expression of CD39 and CD8 increased significantly in tumor tissues compared to paired peritumor tissues. The expression of tumoral CD39 (tCD39), but not tumoral CD8 (tCD8), was related to overall survival. Furthermore, the CD39(+)/CD8(+) ratio was associated with poor prognosis in resected GC patients. Taken together, our data indicate that highCD39 expression and high tCD39(+)/CD8(+) ratio in GC is a predictor of poor prognosis for GC patients after radical resection. Moreover, CD39 could serve as a potential target for cancer immunotherapy.

Ampulla dilation with different sized balloons to remove common bile duct stones.

AIM: To assess the outcomes of ampulla dilation with different sized balloons to remove common bile duct (CBD) stones. METHODS: Patients (n = 208) were divided into five groups based on the largest CBD stone size of < 5, 6-8, 8-12, 12-14, and > 14 mm. Patients underwent limited endoscopic sphincterotomy (EST) alone or limited EST followed by endoscopic papillary balloon dilation with 8, 10, 12 and 14 mm balloons, such that the size of each balloon did not exceed the size of the CBD. Short- and long-term outcomes, such as post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis, perforation, bleeding, and pneumobilia were compared among the five groups. RESULTS: The overall rate of successful stone removal in all groups was 100%, and all patients were cured. Eight (3.85%) patients had post-ERCP pancreatitis, none had perforations, and 6 (2.9%) had bleeding requiring transfusion. There were no significant differences in early complication rates among the five groups. We observed significant correlations between increased balloon size and the short- and long-term rates of post-ERCP pneumobilia. Post-ERCP pancreatitis and bleeding correlated significantly with age, with post-ERCP pancreatitis occurring more frequently in patients aged < 60 years, and bleeding occurring more frequently in patients aged > 70 years. We observed a significant correlation between patient age and the diameter of the largest CBD stone, with stones > 12 mm occurring more frequently in patients > 60 years old. CONCLUSION: Choosing a balloon size based on the largest stone diameter is safe and effective for removing CBD stones. Balloon size should not exceed 15 mm.

Dynamic activity of NF-kappaB in multiple trauma patients and protective effects of ulinastain.

OBJECTIVE: To investigate the dynamic activity of NF-kappaB at the early stage of injury in multiple trauma patients and the protective effects of ulinastain. METHODS: From January 2008 to May 2010, patients with multiple traumas admitted to our emergency department were enrolled in this study. Their age varied from 20-55 years. All enrolled patients were assigned randomly into control group (26 cases of multiple injury without ulinastain treatment), ulinastain group (25 cases of multiple injury with ulinastain treatment), and mild injury group (20 cases) for basic control. The inclusion criteria for mild injury group were AIS-2005 less than or equal to 3, single wound, previously healthy inhospital patients without the history of surgical intervention. In addition to routine treatment, patients in ulinastain group were intravenously injected 200 000 IU of ulinastain dissolved in 100 ml of normal saline within 12 hours after injury and subsequently injected at the interval of every 8 hours for 7 days. NF-kappaB activity in monocytes and the level of TNF-alpha,IL-1, IL-6 in serum on admission (day 0), day 1, 2, 3, 4, and 7 were measured. Data were compared and analyzed between different groups. RESULTS: NF-kappaB activity in monocytes and TNF-alpha,IL-1 and IL-6 of these patients reached peak levels at 24 hour after trauma, with gradual decrease to normal at 72 hour after trauma. NF-kappaB activity and levels of TNF-alpha,IL-1 and IL-6 were lower in ulinastain group than control one, without any significant difference between the two groups. The mean duration for systemic inflammatory response syndrome and multiple organ dysfunction syndrome was 7 d+/-3.1 d and 10 d+/-3.5 d in ulinastain group and control group respectively, and showed a significant difference. CONCLUSIONS: NF-kappaB activity in monocytes and the levels of inflammatory cytokines in multiply injured patients increased transiently at the early stage of trauma. Ulinastain may shorten the duration of systemic inflammatory response syndrome and multiple organ dysfunction syndrome, but does not show the ability to decrease the activity of NF-kappaB .

Comparison of severe trauma care effect before and after advanced trauma life support training.

OBJECTIVE: To study the emergency care effect of in-hospital severe trauma patients with the injury severity score (ISS) larger than or equal to 16 after medical staff received advanced trauma life support (ATLS) training. METHODS: ATLS training was implemented by lectures, scenarios, field practices, and examinations. The clinical effect of in-hospital severe trauma care was compared 2 years before and after ATLS training. RESULTS: During 2 years (from January 1, 2004, to December 31, 2005) before ATLS training, 438 cases of severe trauma were admitted and treated emergently in our department. Among them, ISS score was 28.6+/-7.8 on average, and 87 cases died with the mortality of 19.9%. The duration in emergency department and from admission to operation were 69.5 min+/-11.5 min and 89.6 min+/-9.3 min respectively. Two years (from January 1, 2007, to December 31, 2008) after ATLS training, 382 cases of severe trauma were admitted and treated. The ISS was 25.3+/-6.1 on average and 62 cases died with the mortality of 15.1%. The duration in emergency department and from admission to operation were 47.8 min+/-10.7 min and 61.5 min+/-9.9 min respectively. The ISS score showed no significant difference between the two groups (P > 0.05), but the mortality, the duration in emergency department and from admission to operation were markedly decreased after ATLS training and showed significant difference between the two groups (P <0.05). CONCLUSION: ATLS course training can improve the emergency care effect of in-hospital severe trauma patients, and should be put into practice as soon as possible in China.

First aid strategy for severe traumatic patients in hospital.

OBJECTIVE: To study the emergency management principles of severe trauma in hospital (injury severity score larger than or equal to 16). METHODS: We used "ATP principle" to manage severe traumatic patients. The ATP principle is composed of: 1) attending surgeons offering initial management (A); 2) teamwork commencement immediately after patients admitted to hospital (T); 3) parallel principle, ie, emergency resuscitation, evaluation and laboratory test performed simultaneously (P). Clinical effects before and after applying ATP principle were retrospectively analyzed and compared. RESULTS: During January 1, 2002 to December 31, 2003, 338 patients were treated without applying ATP principle, in which ISS was 25.9+/-6.4, 152 cases died with the mortality being 39.2%, and the time stayed in emergency department and the time to operation room after admission were (102.8+/-16.7) min, (140.3+/-20.6) min, respectively. During January 1, 2004 to December 31, 2005, 438 patients were treated based on ATP principle, in which ISS was 28.6+/-7.8, 87 cases died with the mortality being 19.9%, and the time in emergency department and the time to operation room after admission were (69.5+/-11.5) min, (89.6+/-9.3) min, respectively. ISS showed no significant difference between the two groups (P larger than 0.05) but the mortality, the time stayed in emergency department and the time to operation room after admission were greatly reduced and showed significant difference between the two groups (P less than 0.05). CONCLUSIONS: Applying ATP principle to treat severe traumatic patients can shorten emergency treatment time in hospital and decrease mortality.

Duodenoscopy in treatment of acute gallstone pancreatitis.

OBJECTIVE: To probe the potential use of duodenoscopy in the diagnosis and treatment of acute gallstone pancreatitis (GP). METHODS: Fourty-five patients with acute GP were randomly divided into endoscopic retrograde cholangiopancreatography (ERCP) group (n=20) and non-ERCP group (n=25). Each group was subdivided into mild and severe groups according to APACHE II scores. They were given supportive treatment combined with traditional Chinese medicine. The patients in the ERCP group received ERCP within 24 hours after admission. If there were stones in the common bile duct with stenosis of the inferior extremity or ampulla, endoscopic sphincterotomy (ES) was performed to extract the stones by basket. If no calculi were identified or multiple stones were large, endoscopic naso-biliary drainage (ENBD) was carried out. RESULTS: The incidence of complication, length of hospitalization and cost were markedly lower in patients with severe acute GP in the ERCP group than those in the non-ERCP group (P<0.05), in contrast to the 2 mild subgroups of the ERCP and non-ERCP groups (P>0.05). CONCLUSION: It is feasible, effective and safe to apply duodenoscopy in the treatment of severe acute GP.