This work was to evaluate the perioperative safety and efficacy of cytoreductive surgery (CRS) plus hyperthermic intraperitoneal chemotherapy (HIPEC) with lobaplatin and docetaxel in patients with peritoneal carcinomatosis (PC) from gastrointestinal and gynecological cancers.2 and docetaxel 60 mg/m2 in 6000 mL of normal saline at 43 ± 0.5 °C for 60 min. Vital signs were recorded for 6 days after CRS + HIPEC procedures. Perioperative serious adverse events (SAE), hematological, hepatic, renal, and electrolytes parameters, the changes in serum tumor markers (TM) before and after operation, patient recovery, and overall survival (OS) were analyzed.Patients were treated by CRS + HIPEC with lobaplatin 50 mg/mOne hundred consecutive PC patients underwent 105 CRS + HIPEC procedures and postoperative chemotherapy. The median CRS + HIPEC duration was 463  min, and the highest temperature and heart rate during six postoperative days were 38.6 °C (median 37.5 °C) and 124 bpm (median 100 bpm), respectively. The 30-day perioperative SAE occurred in 16 (15.2 %) and mortality occurred in 2 (1.9 %) patients. Most routine blood laboratory tests at 1 week after surgery turned normal. Among 82 cases with increased preoperative TM CEA, CA125, and CA199, 71 cases had TM levels reduced or turned normal. Median time to nasogastric tube removal was 5  days, to liquid food intake 6  days, and to abdominal suture removal 15  days. At the median follow-up of 19.7  months, the median OS was 24.2  months, and the 1-, 3-, and 5-year OS rates were 77.5, 32.5, and 19.8 %, respectively. Univariate analysis identified five independent prognostic factors on OS: the origin of PC, peritoneal cancer index, completeness of CRS, cycles of adjuvant chemotherapy, and SAE.CRS + HIPEC with lobaplatin and docetaxel to treat PC is a feasible procedure with acceptable safety and can prolong the survival in selected patients with PC.NCT00454519ClinicalTrials.gov,  The locoregional progression of gastrointestinal and gynecological malignancies such as gastric cancer (GC), colorectal cancer (CRC), ovarian cancer (OC), primary peritoneum carcinomatosis (PPC), pseudomyxoma peritonei (PMP), malignant peritoneal malignancies (MPM), frequently results in peritoneal carcinomatosis (PC), characterized by the presence of tumor nodules of various size, number, and distribution on the peritoneal surface, with significant negative impact on both survival and quality of life, due to refractory ascites, progressive intestinal obstruction, and unrelieved abdominal pain. The traditional therapies for PC include systemic chemotherapy, palliative surgery, and best support care, without any hope of cure. PC patients have a very poor prognosis with median survival less than 6 months , 2.With better understanding of the tumor biological behaviors and advances in treatment technologies, the landscape of PC has changed remarkably, in that PC is regarded as locoregional disease rather than widespread terminal condition. Over the past decades, novel therapeutic approaches to PC have emerged, combining cytoreductive surgery (CRS) plus hyperthermic intraperitoneal chemotherapy (HIPEC), which integrate the advantages of surgically removing the bulky visible tumor burden and regional hyperthermic chemotherapy eradicating micrometastases and invisible free cancer cells .Hyperthermia and chemotherapy have synergistic effects, which is significantly enhanced at 42 °C . The chePhase I/II clinical trials of lobaplatin in combination with docetaxel have demonstrated acceptable safety and efficacy for a variety of malignant solid tumors , 14. SevThis study included 100 consecutive patients of PC treated by 105 CRS + HIPEC procedures from January 2008 to June 2015, including 41 patients from CRC, 30 from GC, 16 from OC and PPC, and 13 from PMP. Major clinicopathological characteristics of the patients are listed in Table A designated team of surgical oncologists, anesthesiologists, and operating room staff focusing on PC therapy performed CRS + HIPEC procedure, with detailed description reported previously , 16. Bef2 of lobaplatin and 60 mg/m2 of docetaxel each dissolved in 3 L of saline. When the perfusion saline was kept at 43.0 ± 0.5 °C and monitored with temperature sensors on real time by an automatic hyperthermia chemotherapy perfusion device , the perfusion solution was infused at a rate of 400 mL/min. The total HIPEC time was 60 min. After HIPEC, the perfusion solution in the peritoneal cavity was withdrawn by the suction. Then the operation field was checked again for any suspected mishaps. The anastomoses were made to restore the continuity of the digestive tract. Routine colostomy was not performed, but in patients with extensive pelvic resections of the sigmoid colon, rectum, uterus, and bladder, colostomy was made. Then drainage tubes were placed at appropriate sites of anastomosis and depending on the type of primary operation. The incision was closed with a tension-reduced suture, and patient was transferred to the intensive care unit (ICU) for recovery.After CRS, abdominal cavity was sufficiently open by Mediflex Surgical Products and all-layer surgical incision protector covered the open abdomen in order to keep the temperature stable and avoid chemotherapy drugs diffusing into the air before HIPEC, because the open coliseum technique was thought to provide optimal thermal homogeneity and spatial diffusion , with 50All patients were carefully monitored for the following parameters: (1) The changes in body temperature and heart rate were recorded for 6 days after surgery; (2) The complete peripheral blood routine, blood biochemical parameters were examined on the first and seventh postoperative days, and cardio-pulmonary function was monitored, the change in serum tumor markers (TM) before and after operation; (3) Anastomotic leakage, intra-abdominal infection, hemorrhage, intestinal obstruction, and other life-threatening serious adverse events (SAE); (4) Wound healing, including fat liquefaction, incision split, surgical site infection, incisional hernia, time to suture, and drainage tube removal; (5) Postoperative recovery of gastrointestinal and other organs functions, including bowel sound, flatus passage, defecation, time of removing nasogastric tube and urinary catheter, and liquid food intake time; and (6) Survival outcomes.2, on day 1, every 21 days) and docetaxel , and systemic chemotherapy (SC) mainly with irinotecan, leucovorin, and 5-fluorouracil (FOLFIRI) and oxaliplatin, leucovorin, and 5-fluorouracil (FOLFOX) regimens, all dosed according to body surface area calculation [Postoperative adjuvant chemotherapy included intraperitoneal chemotherapy (IP) through the IP port mostly using lobaplatin  every 3 months during the first 2 years and then every 6 months thereafter by outpatient consultation or by telephone. The last follow-up was on June 8, 2015, and the overall follow-up rate was 100 %.P < 0.05 considered as statistically significant. The numerical data were directly recorded, and the category data were recorded into different categories.All data were obtained from a prospectively established database of clinical records, surgical and pathology reports, image examination and laboratory reports, and follow-up records. And data were analyzed by SPSS software for windows, version 19.0 , with The following study parameters were defined: (1) Perioperative period: from the day of CRS + HIPEC to day 30 postoperation; (2) Metachronous PC: after the primary tumor had been treated, the patients developed PC during follow-up; (3) Synchronous PC: PC was diagnosed synchronously at first treatment; (4) PCI ≤20 was defined as low PCI (LPCI), and PCI >20 was defined as high PCI (HPCI) ; (5) TheA total of 100 PC patients were treated with 105 CRS + HIPEC procedures, including five patients each receiving two CRS + HIPEC procedures . There were 59 women and 41 men, with a median age of 51  years. The median operation time was 463  min. During surgery, the median volume of blood loss, ascites, and urine output were 600  mL, 500  mL, and 1200  mL, respectively, and the median transfusion volume of red blood cells, plasma, cryoprecipitation and other fluids were 600  mL, 600  mL, 150  mL, and 3500  mL, respectively. In terms of organ resection, 59 patients received removal of 1-3 organs segments, 23 patients had excisions involving 4-7 organs segments, and 6 patients had resections involving 8–12 organs segments. All patients underwent peritonectomy in more than one area of the peritoneal and pelvic cavity  days, to nasogastric tube removal 5  days, to fluid food intake 6  days, to drainage tube removal 9  days, to abdominal sutures removal 15  days, and to hospital discharge 20  days.n = 4), intestinal obstruction (n = 2), acute myocardium infraction (n = 1), and declining any chemotherapy (n = 12).After operation, 30 patients received a median of 5  cycles of SC and 51 patients received a median of 6  cycles of PIC + SC procedures. The other patients did not receive adjuvant chemotherapy due to intestinal leakage   vs. 16.3  months   vs. 13.6  months   months vs. 14.1  months   months vs. 12.2  months   months for all PC patients in this study, there were 55 (55 %) patients who died, 16 (16 %) survived with tumor, and 29 (29 %) survived without tumor. The 1-, 3-, 5-year OS rates were 77.5, 32.5, and 19.8 %, respectively. The median OS was 24.2  months Fig. . The OS st) Fig. , respectst) Fig. . The medst) Fig. . The medst) Fig. . The medst) Fig. .Fig. 1ThStaphylococcus aureus and Candida tropicalis infection, with flushing abdominal cavity, intraperitoneal drainage, antibiotics, and total parenteral nutrition support, the patient survived 2.3 months after the operation. The third patient with generalized peritonitis syndrome on postoperative day 11 then developed septicemia due to gram-negative bacilli; given the above active conservative treatment, the patient survived 24 days after the surgery. The fourth patient developed late-onset mild anastomosis fistula on postoperative day 30 and received conservative treatment; the patient survived 3 months after the surgery.SAE grades 3–5) occurred in 16 (15.2 %) of 105 CRS + HIPEC procedures  on postoperative days 6 and 8, respectively, received antidiarrheal therapy, restoration of intestinal flora and electrolytes supplementation therapy, and recovered after 15 and 20 days, respectively. Four patients developed septicemia, two of whom were secondary to above anastomosis leakage, and the other two patients were infected with vs. 12.6 months with standard palliative therapy) and recommends that CRS + HIPEC be the standard treatment model for CRC PC patients [vs. 13.4 months) [CRS + HIPEC as a comprehensive treatment strategy makes the best of surgical resection, locoregional chemotherapy, hyperthermal therapy, and large volume abdominal perfusion washing by CRS to remove the peritoneal and abdominopelvic visible tumor, and the synergistic effects of HIPEC to eradicate residual tumor nodules, micrometastases, and free cancer cells. So far, it is the most effective strategy to treat PC . We havepatients . Spiliot months) .The peritoneal-plasma barrier limits the absorption of large molecule-weight drugs by the peritoneum, ensuring high concentration of drugs in the abdominal perfusion solution. As a result, HIPEC increases the direct cytotoxic effects of chemotherapeutic drugs on peritoneal surface tumors and reduces the systemic side effects. The mechanism of lobaplatin is similar to that of other platinum drugs. Specifically, lobaplatin induces the formation of inter-strand Pt-GG and Pt-AG crosslinks, hindering DNA replication and transcription ultimately inhibiting gene expression in tumor cells . Lobapla2 + docetaxel 60 mg/m2 for 60 min); (3) tension-reduced suture in closing abdominal incision and abdominal wound caring every day to ensure proper wound healing; (4) transfusion of blood, fresh-frozen plasma, cryoprecipitation, and albumin to promptly restore hemostasis; (5) use effective broad-spectrum antibiotics to cover gram-positive, gram-negative, anaerobic, and aerobic bacteria; (6) postoperative ICU monitoring; and (7) good nutritional support and quick restoration of electrolytes balance.To achieve maximal CRS including organs and peritoneal areas resection, the field of operation should be wide, increasing the risks for major complications. A single-center large sample-size study on 243 PC patients revealed a mortality rate 3 %, SAE rate 43 %, and the SAEs mostly being intra-abdominal collection (40 %), infection (38 %), pleural effusion (37 %), pneumothorax (15.6 %), fistula (11.5 %), ileus (9.5 %), pneumonia (7.4 %), bleeding (5.8 %), perforated viscus (5.8 %), pancreas leak (4.5 %), cardiac arrhythmias (4.1 %), and pulmonary embolism (2.9 %) . In thisDetailed analysis of the 16 SAEs reveals the following features: (1) more advanced clinical stages, with median PCI 26 ; (2) more complex and difficult operation procedures, with median organ resections, areas of peritonectomy, CC, and operation time being 4 , 8 , 2 , and 660 min , respectively; (3) significantly greater negative impact of SAEs on OS, with median OS of only 12.2  months for SAEs vs. 31.2  months for non-SAEs. Therefore, more attention should be paid to selecting LPCI patients for CRS + HIPEC treatment, and intensified perioperative care to minimize the risk of SAEs.In addition to routine hematological examination, the detection of serum TM was necessary. In this study, 82 patients had elevated serum CEA, CA125, or CA199 levels before operation, and 71 cases (86.6 %) had postoperative TM levels reduced or returned to normal. As serum CEA, CA125, and CA199 levels could reflect tumor invasiveness , peritonThe median OS for the 100 patients was 24.2  months, with the 1-, 3-, and 5-year survival rates being 77.5, 32.5, and 19.8 %, respectively. From this study, several lines of evidence could be obtained to support the comprehensive strategy for PC. First, the median disease specific OS could reach 31.2 and 27.5 months for patients with non-neoadjuvant chemotherapy and synchronous PC, but 20.7 and 13.2 months for those with neoadjuvant chemotherapy and metachronous PC. Although such differences did not reach statistical significance, possibly due to follow-up time and sample size, the data do suggest that non-neoadjuvant chemotherapy and synchronous PC could obtain relatively better survival from the therapy. Second, in terms of origin of PC, the PMP and OC PC achieved better OS than the others. In our study, the median OS of OC PC, GC PC, and CRC PC was 34.6, 15.7, and 14.9 months, respectively, similar to the literature reports of 26.7, 12.0, and 13.7 months , 24, res2) combined with docetaxel (60 mg/m2) to treat human solid tumors  could exhibit short-term efficacy  and a low incidence of SAE, such as leukopenia 30 % (grade 3), neutropenia 50 % (grades 3–4) without non-hematological toxicities, with median chemotherapy cycles of four  [2 combined with docetaxel 60 mg/m2, and this regimen demonstrated broad-spectrum anti-tumor properties against lung cancer, breast cancer, endometrial cancer, and gastric cancer. A phase II study evaluated the clinical efficacy and safety of lobaplatin (30 mg/m2) combined with docetaxel (75 mg/m2) on 39 patients with recurrent or metastatic nasopharygenal carcinoma [2) used in intrapleural or intraperitoneal infusion is a safe and effective treatment for patients with malignant pleural effusion or ascites from colorectal cancer or uterine cancer [2 of lobaplatin and 60 mg/m2 of docetaxel) used is comparable to those reported above. And the adverse events were also comparable to the reported results.To our knowledge, there have been three major publications on the combination of lobaplatin and docetaxel in clinical setting , 14, 34. cycles) . This phCRS + HIPEC with lobaplatin and docetaxel to treat PC is a feasible procedure with acceptable safety and can prolong the OS of patients with PC from gastrointestinal and gynecological malignancies.


These findings indicate that miRNA-155-5p may be involved in PE pathogenesis and could be a potential biomarker for this disease.It has been well documented that preeclampsia (PE) has a common etiological background, but little is known about its linkage at the molecular level.Non- coding RNAs are critical posttranscriptional regulators ofgene expression. This study was performed to determine whether PE is associated with alterations in placental non-coding RNAs expression. MicroRNA (miR)-155-5p and long non-coding RNA ( Accordiet genes . Mir- 15et genes . Evidencpression , 11. MorNOS mRNA . Since tof eNOS3 . Given tStudy population>140mmHg and /or diastolic bloodpressure> 90mmHg on at least two occasionsafter gestational week 20), with proteinuria (>0.3 g/24 h).Severe PE was defined as blood pressure above 160 mmHg systolic and/or 110 mmHg diastolic or proteinuria >5 g/24 h or oliguria (<500 ml urine/24 h). All pregnancies were otherwise uncomplicated singleton pregnancies except for the development of PE. Patients with chronic hypertension, renaldisease, collagen vascular disease,a positive family history for PE and otherpregnancy complications  were excluded from this study. The EthicsCommittee of ShahidBeheshti University of Medical Sciences approved the consentforms and procedures necessary to utilizethe tissues, and written informed consentwas obtained from all participants prior tosurgery.Placental tissue samples were sequentially collectedfrom 59 Iraniannulliparous pre-eclamptic womenwithout family history of disease, and 40 gestational age-matched nulliparous normotensive pregnancies (controls) at the Department ofObstetrics and Gynaecology, Mahdiyeh hospital, of ShahidBeheshti University of Medical Sciences, Tehran, Iran during March 2014 and July 2015 after delivery.The average of pre-eclamptic women and control group who delivered by caesarian sections were 70% and 12.5%, respectively. PE was defined according to the American College of Obstetricians andGynecologists guidelines as gestational hypertension  and kept at −80 °C until RNA isolation. All samples were collected by the same medical personnel and using identical protocol.RNA extractionRNA was extracted using the miRNeasy Mini Kit  according to manufacturer’s instructions. RNA quality was assessed by agarose gel electrophoresis. Only samples with two clearly detectable ribosomal RNA bands (28S and 18S) and a higher than 2:1 ratio-were evaluated by nanodrop for purityof intact RNA, and were used for reverse transcription.cDNA synthesis and quantitative real- time reverse transcription (RT) PCR for miR-155 expression analysis,s formula (2^-Δct).Real-time quantitative PCR was performe-dusing a Corrbetreal-time PCR detection system , Qiagen, Germany). Each 15μl reaction volume contained7.5 µl of master mix , 5 µl nuclease free water, 1µl offorward primer plus universal reverse and1.5µl cDNA. PCRamplifi-cation of mir- 155 was initiated at 95°C for 5 min, followed by 45 cycles of 95°C for15 s, 61°C for 45s.Expression of miR-155was assessed byquant-itative real-time RT–PCR(SYBR green method), and thesmall nuclear RNA U6was used as aninternal control. The miR-155 and RNAU6-specific cDNA sequences were synthesizedfrom total RNA using gene-specificprimers . cDNA sycDNA synthesis and quantitative real-time reverse transcription (RT) PCR for lncone expression analysislnconeRNA was analyzed by converting 5 µl RNA sample to cDNA for each sample, using an RNAreverse transcription kit with random hexamer and oligo dTprimers , according to the manufacturer’s instructions. Briefly, reagents were prepared to obtain a final concentration of 1 µl random hexamerprimer,1 µl oligo dTprimer, 5 µl template RNA, 5 µl nuclease free water, 1µl RiboLock RNase Inhibitor (20 U/μl), 2 µl dNTPs (10 mM) and 1 µl RevertAid M-MuLV RT (200 U/μl) in a finalvolume of 20 µl.The RT reaction was performedby incubating the samples for 5 minat 25°C, 1 h at 42°C and 5 min at 70°C. Real-time quantitative PCR was performed using Corbet Real-Time PCR System (Rotor gene Corbett 6000(2.1.0), Qiagen, Germany) and SYBR_qPCR Mix (Takara). After performing gradient PCR to find the appropriate temperature . Eacc 5μl reaction volume contained 7.5 µl master mix , 5 µl nuclease free water, 1µl forward and reverseprimers .Statistical analyzes were performed using SPSS 21.0 for Windows  and Graph Pad Prism 5.0 . Data are expressed as mean± SD or as mean ± SEM where appropriate. Comparisons of the groups were examined by Mann–Whitney U test for nonparmetric data. A P value <0.05 was considered as statistically significant for all analyzes.The threshold cycle (Ct) was defined as the fractional cycle number at which Fluorescence passed the fixed threshold. Each sample was measured in duplicate, and the relative amount of miR-155 to U6 and PatientsIn this study the average age and body mass index (BMI) in PE woman were: 27.42 ± 6.7 years, and 23.87 ± 4.15, respectively, and 25.90 ± 3.96 years, and 23.33 ± 2.53, respectively for control group. No significant differences were identified between the normal pregnant and the PE patients with respect to maternal age, but the P- value for BMI was significant (P<0.001) .Placental expression of miR-155-5p and lnconeIncreases in miR-155-5p expression in placental tissue from patients with PE  was 1.6 fold and was found to be significantly higher than the normotensive control group  was 1.1 fold and was not significantly different from the normotensive control group  in PE placenta, which may be an important pathway in the development of PE. Moreover, miR-155, as a common target of a wide range of inflammatory mediators, can be up- regulated by different inflammatory factors, including LPS, TNF and IL-1 through affecting Toll like receptors (TLR)  . In the lncone was sligthly increased. However, this increase was not statistically significant. In 2004, Robb et al. evaluated the expression of sONE in human umbilical vein endothelial cells (HUVEC). They found that the expression level of sONE was increased in vascular smooth muscle cells upon knock-down of sONE by siRNA (sONE was reduced in endothelial cells, in different types of cells under the intense hypoxic condition, the expression level of this mRNA increased significantly. In endothelial cells and normal oxygen condition, a complex of proteins stabilized the eNOSmRNA through binding to the eNOS 3'UTR (eNOS and sONE compete with each other for binding to RNA stabilizing proteins or sONE/eNOS RNA interaction prevents the formation of eNOS 3'UTR protective complex (sONE was evaluated in a case–control study through collection of PE and normal placenta tissues.In our primary analysis, we found that the expression level of by siRNA . MoreoveOS 3'UTR . It is b complex -20. ThesOverall, our results suggest that perturbations in NO production and NO- related genes such as mir-155-5p may have a tight relation to the pathogenesis of PE. However, further studies are needed to confirm this fact. Since this study was performed on nulliparous women, the results obtained from our investigation could not be applied to all pregnant women. In some cases, probably using other hypothesesfor the pathogenesis of the disease could provide a comprehensive description for the risk factors associated with PE, but proving this requires further researches.Based on the remarkable role in disease pathogenesis, designing drugs targeting NO production pathway, NO-related genes and biological reinforcing of NO could be a significant therapeutic strategy for patients. Moreover, further studies are warranted on NO production pathway and NO-related genes to more precisely characterize the molecular mechanisms involved in this disease.


Extracellular vesicles (EVs) are membrane bound, secreted by cells, and detected in bodily fluids, including urine, and contain proteins, RNA, and DNA. Our goal was to identify HIV and human proteins (HPs) in urinary EVs from HIV+ patients and compare them to HIV− samples.n = 35) and HIV− (n = 12) individuals. EVs were isolated by ultrafiltration and characterized using transmission electron microscopy, tandem mass spectrometry (LC/MS/MS), and nanoparticle tracking analysis (NTA). Western blots confirmed the presence of HIV proteins. Gene ontology (GO) analysis was performed using FunRich and HIV Human Interaction database (HHID). Urine samples were collected from HIV+ (P < 0.05, by NTA. HIV+ samples had 14,475 HPs using LC/MS/MS, while only 111 were in HIV−. HPs in the EVs were of exosomal origin. LC/MS/MS showed all HIV+ samples contained at least one HIV protein. GO analysis showed differences in proteins between HIV+ and HIV− samples and more than 50% of the published HPs in the HHID interacted with EV HIV proteins. EVs from urine were 30–400 nm in size. More EVs were in HIV+ patients,  Differences in the proteomic profile of EVs from HIV+ versus HIV− samples were found. HIV and HPs in EVs could be used to detect infection and/or diagnose HIV disease syndromes.  These Egression  that aregression  in HIV igression , other vgression , and othgression –11.EVs from urine are an attractive noninvasive source for biomarkers of diseases , 13. In  in vitro transfected or HIV infected cultured cells and are not from HIV+ patient samples [HIV proteins are detected in EVs of HIV+ patients and HIV Nef is the most prevalent protein found –21. Othe samples , 22, 23.Biomarkers in urinary EVs are suggested for use in the diagnosis of many disease states , 24–30. Urine was collected from thirty-five (35) HIV+ patients and twelve (12) HIV− individuals in sterile collection cups. The subjects were recruited from clinics in the metropolitan Atlanta area, GA. Patient demographics are described in μl phosphate buffered saline (PBS) for use in the transmission electron microscopy and tandem mass spectrometry (LC/MS/MS) analysis.Urine samples were centrifuged at 1000 ×g to remove cells and sediment then frozen at −80°C. Samples, 4 ml, were thawed and the EVs isolated followed by centrifugal filtration using Amicon Ultra-4 100 kDa centrifugal filter unit , at 3000 ×g for 15 minutes at 4°C. The retentate, containing EVs, was collected from the top of the filter and resuspended in 200 Transmission electron microscopy (TEM) was used to identify EVs in two HIV-1 positive and two HIV-1 negative samples. Urinary EVs were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 2 hours at 4°C followed by 2 washes with 0.1 M cacodylate buffer, 5 minutes each. Samples were stained with 1% osmium tetroxide in 0.1 M cacodylate buffer for 1 hour at 4°C followed by 2 washes with the cacodylate buffer and 3 washes with deionized water, 5 minutes each. Samples were subsequently stained with 0.5% aqueous uranyl acetate for 2 hours at room temperature and subsequently viewed with a JEOL 1200EX transmission electron microscope .n = 8) and positive individuals (n = 11), 15 ml, were centrifuged at 300 ×g for 10 min at 4°C to remove cell debris. The supernatant was collected and centrifuged at 16,500 ×g for 20 min at 4°C and the supernatant collected and ultracentrifuged at 120,000 ×g at 4°C for 1.5 hr. The pellet was resuspended in 500 μl of PBS. The size and quantification of the EVs were analyzed using the NanoSight NS500 . Particles were automatically tracked and sized based on Brownian motion and the diffusion coefficient. The NTA measurement conditions were temperature 21.0 +/− 0.5°C, viscosity 0.99 +/− 0.01 cP, frames per second 24.99–25, and measurement time 30 s. The detection threshold was similar in all samples. Two recordings were performed for each sample.Urine samples from HIV-negative  on a 0.5 × 75 mm C-18 column (Michrom) at a flow rate of 500 nl/min using a linear gradient of acetonitrile (5–35%) over 100 min. Ions were directly introduced by nanospray and spectra were collected using Xcalibur 2.0 software using an intensity threshold of 200 counts. The resulting spectra were analyzed using Bioworks 1.1 software to search a hybrid Human-HIV database created from the complete nonredundant peptide database from NCBI. The threshold for inclusion in the search is a minimal S/N ratio of 3. False discovery rates were determined and set based on the control HIV− samples. An initial protein identification list was generated from matches with an Xcorr score versus charge state of 1.0 (+1) 1.5 (+2) and 1.7 (+3) and consensus scores greater than 10.0.Thirty-five (35) HIV+ and twelve (12) HIV− EV samples were lysed and trypsinized and the sequence of peptides was determined by tandem mass spectrometry (LC/MS/MS), using an LTQ Ion Trap Mass Spectrometer . Peptides were first reduced in DTT 10 mM at 56°C for at least 30 min and alkylated with 15 mM iodoacetic acid for 30 min at room temperature in the dark. Samples were then digested with mass spectrometry grade trypsin 20 ng/http://funrich.org/index.html) [P value < 0.05, using the Benjamini-Hochberg False Discovery rate, were reported. The human proteins detected were compared to the top 100 EV proteins in ExoCarta (http://exocarta.org/exosome_markers_new) [Bioinformatics techniques for analysis of HIV EV proteins were used on the LC/MS/MS detected proteins . Functioex.html)  against ers_new) , 34, sixers_new) , and proers_new) .n = 15) to those with less than 500 (n = 15) and HIV high VL, greater than 200 copies (n = 10), compared to HIV low viral loads, less than 200 copies (n = 10), was done using Pathway Studio version 11.4 Mammal Plus . Gene Set Enrichment Analysis (GSEA) was used to identify the top 10 curated pathways for the proteins in the each of the patient groups. No comparisons were done between patients not on ART or undergoing ART because there was only one patient not on ART.Pathway analysis comparing HIV+ samples with CD4+ T cells greater than 300 (https://www.ncbi.nlm.nih.gov/genome/viruses/retroviruses/hiv-1/interactions/). This database contains all the known, published interactions of HIV-1 gene products with human proteins [The HIV proteins, Nef, Vpr, Vpu, and Vif, were searched using the HIV-1 Human Interaction database  was performed on twenty (20) randomly selected HIV+ and three (3) HIV− control urine samples. Recombinant HIV-1 Nef and HIV-1 p24 were used as positive controls, while HIV-negative urine and HIV-positive filtrate were used as negative controls. Samples were heated at 85°C for two minutes in a tris-glycine SDS sample buffer, were loaded into a 4–20% TGX gradient gel , and run for 50 mins at 200 V. A semidry transfer unit  was used to transfer the separated proteins onto a PVDF membrane (Bio-Rad) at 15 V for 50 mins. The filter was blocked for nonspecific binding using 5% nonfat dry milk in 1x tris buffered saline (TBS) with Tween 20. The membrane was incubated overnight in pooled plasma from twenty HIV+ patients as the primary antibody at a 1 : 500 dilution and rabbit anti-human IgG conjugated HRP antibody  was used as secondary antibody. Super Signal West Femto  was used as a chemiluminescent substrate for detection. The membrane was developed and imaged using the LAS 4000 biomolecular imager . Recombinant HIV-1 Nef and p24 WB analyses were detected using anti-Nef and p24 monoclonal antibodies  and anti-mouse IgG conjugated HRP antibodies  were used.Twenty-six (26) HIV+ and eleven (11) HIV− urine samples were tested for the presence of HIV p24 by ELISA .n = 35) contained at least one HIV-1 protein in EVs, while no HIV proteins were found in the HIV− samples (n = 12) (LC/MS/MS mass spectrometry HIV EV protein results are presented in (n = 12) . HIV-1 NHIV p24 antigen was only detected in five of thirty-five (5 of the 35 patient) (14%) samples by LC/MS/MS, but of the twenty-six (26) HIV+ and eleven (11) HIV-negative samples tested by ELISA, no p24 was detected. There was no statistical correlation of the number of HIV proteins detected with CD4+ T cell counts, viral loads, or ART therapy.Validation by WB analysis using polyclonal pooled patient serum and monoclonal antibodies against HIV Nef and HIV p24 indicated the presence of HIV proteins. P < 0.05). No significant differences were found in the size of the EVs, 110–227 nm for HIV-negative donors and 54–448 nm HIV+ samples. Representative Nanosight analyses for HIV-negative and HIV+ urine samples are shown in TEM analysis of urine from HIV+ patients showed multiple EVs, ranging in size from 50 nm to 300 nm , while thttp://exocarta.org/exosome_markers_new) [EV proteins from the HIV+ patients, 14,475, which entered into FunRich, functional enrichment analysis software, showed 29.44% or 1,932 proteins were associated with exosomes . These Eers_new) , 22 EV pers_new)  were simers_new)  were fouP < 0.01) EV sites of expression were endothelial cells, plasma, liver, serum, and kidney and the most significant cellular components were lysosomes, exosomes, membranes, plasma membranes, the nucleus, and the cytoplasm (P < 0.01) (P < 0.0001), the top biological process was regulation of nucleobase, nucleoside, and nucleic acid , and the most prominent biological pathway was integrin cell surface interactions (P < 0.03).The GO results of the FunRich analysis of the EVs from the HIV+ samples are summarized in  < 0.01) . The top < 0.01)  were proβ1, IDO, Notch1, ADAM17, Rab4, and HGF, were also found by LC/MS/MS in addition to MHC Class I and II antigens.LC/MS/MS identified 15,571 proteins in EVs from HIV+ patients with CD4+ T cells greater than 300, 2,115 from CD4+ T cells less than 300, 15,028 proteins from patients with low VL, and 2486 from patients with high VLs. Pathway analysis was similar between EV proteins from patients with greater than 300 CD4+ T cells and low VLs and different between the low CD4+ T cells and high VLs ; HIV Vpr interacted with 437 EV of 598 human (73.1%); HIV Vif interacted with 162 EV of 310 human (52.2%); and HIV Vpu interacted with 165 EV of 244 human proteins which were found in the HIV+ EVs (67.6%) (see Supplementary P < 0.0001). The most significant ontologies were molecular function of the proteins and defense/immunity protein activity and principal biological processes were immune response, signal transduction, cell communication, and antigen presentation (P < 0.0073).Functional analysis of the control EVs are listed in P < 0.001, Only sixty-four (64) proteins overlapped between the HIV+ and control EV samples and are listed in  in vitro HIV infected cell cultures and not from HIV+ patients [This is the first report of the detection of urinary EVs containing HIV and human proteins from HIV+ patients by mass spectrometry and western blot. EVs provide intercellular communication to cells through the delivery of their cargo, nucleic acids, miRNAs, and proteins, to recipient cells reviewed in . Previoupatients , 38–47. According to the International Society for Extracellular Vesicles (ISEV), the minimal requirements for EVs or their presence in samples includes the simultaneous detection of transmembrane proteins and cytosolic proteins with membrane/receptor binding abilities, while major cell organelles are absent . LC/MS/MHIV has previously been detected in the urine of HIV+ patients; however, it was shown that HIV virions are associated with cell pellets and not in centrifuged urine , 51. p24Different proteins and pathways were found in EVs from (1) CD4+ T cell > 300 versus <300 and (2) VLs < 200 versus >200 copies. It is interesting that EVs from HIV+ patients with low VLs and high CD4+ T cells, usually indicative of better health, had more proteins detected than EVs from high VLs and low CD4+ T cells . These groups also had overlapping pathway results; however, proteins from high VLs and low CD4+ T cells did not have similar pathway results. Further comparison and analysis of the EV protein profile between the low VL/high CD4+ T cells and high VL/low CD4+ T cells may reveal more mechanisms involved in the evolving pathology of HIV infection.α and IL1. Both these cytokines were present in the EVs from HIV+ patients while TNF-α and IL1 were not detected suggesting an immunomodulatory effect may be elicited by the EVs. Other immune downregulating factors, IDO, HOXB4, HGF, and TGFβ1, were found. IDO [β-1, an inhibitor of immune function, is induced by HIV Tat [β-1 and IL10 than patients with progressive disease [β-1 and IL10 and remove them from circulation. The presence of over 16 different MHC Class I and II antigens in the EVs from HIV+ patients may support the hypothesis that this mechanism is used by intracellular pathogens to evade the immune response by decreasing cytotoxic T cell activity [Proteins contained in EVs can both enhance and inhibit host responses from innate, inflammatory, and adaptive reactions. Proteins from HIV+ patients showed a predominantly immunosuppressive profile. IL10 is a Th2 cytokine that downregulates macrophage function and inhibits T cell proliferation while IL6 can stimulate IL10 production and inhibit the effects of TNF-und. IDO , HLA-G [und. IDO , and HGFund. IDO  can inhi HIV Tat  and is a HIV Tat –64. Thes disease  and it iactivity . Herpes activity . Future  in vitro and patient samples have been previously reviewed in [In this study, we showed that structural, regulatory, and accessory HIV proteins could be detected in urinary EVs of HIV+ patients. Our WB analysis using polyclonal and monoclonal antibodies confirmed the presence of HIV proteins in the EVs from HIV+ patients. The most prevalent protein was HIV Nef. EVs from bothiewed in . HIV Nefiewed in . WhetherThe HIV Human Interaction database found significant interactions between HIV Nef, Vpr, Vif, and Vpu and human proteins. Serine/threonine protein kinases are important in T cell receptor signaling . These kOne of the limitations of this study was a small sample size of specific HIV syndromes such as comorbidities, AIDS, HIV-associated nephropathy, and HIV-associated dementia as well as patients on or naïve to antiretroviral therapy. Increasing the numbers of HIV+ patients in these categories may allow us to determine whether specific HIV proteins as well as human proteins in urinary EVs could be associated with these conditions. Future studies will also quantify the amount of HIV proteins as well as human proteins to determine if a correlation exists between different HIV conditions and the amount of proteins detected.HIV infection is usually detected by antibodies to HIV and can take up to three months to develop or by measuring VLs in blood whereas we can detect HIV-1 proteins in urinary EVs. In summary, urinary proteins in EVs from HIV+ patients may allow a noninvasive method to (1) rapidly screen for infection and identification of patients eligible for antiretroviral treatment (ART); (2) monitor ART treatment efficacy; and (3) diagnose HIV comorbidities.


Propranolol did not influence IL-6 responses in women, or IL-1Ra in either sex. Blood pressure and heart rate increased markedly during the tasks, but there was no differential stress reactivity in propranolol and placebo conditions. The results of the study support a role of sympathetic nervous system activation in stimulating acute IL-6 responses to stress, but only in men. The reasons for the differences between men and women remain to be resolved.Acute mental stress elicits increases in plasma cytokine concentrations in humans, but the underlying mechanisms remain poorly understood. We assessed the impact of beta-adrenergic blockade on plasma interleukin 6 (IL-6) and IL-1 receptor antagonist (IL-1Ra) responses in a parallel group, double-blind randomised placebo-controlled trial involving 64 healthy young adult volunteers. Participants were administered 80 mg slow-release propranolol or placebo daily for 7 days before the stress testing session in which responses to 3 behavioural challenges  were evaluated. Propranolol administration was associated with reduced baseline levels of heart rate and IL-1Ra, and systolic blood pressure (BP) in men. Tasks stimulated increased plasma IL-6 concentrations sampled 45 min and 75 min after challenge, but these responses were blocked by propranolol in men ( PsychosThe biological mechanisms underlying inflammatory responses to acute stress are only partly understood. Psychological stress elicits rapid increases in expression of nuclear factor κB (NF- κB), a transcription factor promoting the production of IL-6 and IL-1β . IncreasAnother approach to investigating the pathways underlying acute inflammatory responses involves pharmacological blockade . Rodent 22.1Participants were 69 healthy volunteers recruited from the UCL campus for a study assessing the effects of pharmacological probes on stress responsivity. All data were collected with the written informed consent of the participants and ethical approval was obtained from the UCL Research Ethics Committee. Participants were aged 18 years and over, reportedly in good health, and not taking medications regularly (excluding the contraceptive pill). Exclusion criteria included haematological, pulmonary, liver, renal, gastrointestinal, heart, cerebrovascular, and psychiatric disease, history of thromboembolism, and participants were free of current infection. Individuals suffering from asthma, who had known allergies to the study medications, previous gastrointestinal bleedings, or who were currently pregnant or breastfeeding were excluded. Only those with BP in the normal range were included (90/60 mmHg–140/90 mmHg). Three people failed to complete the study, one dropped out because of side effects, and one was excluded after taking cold medication, leaving 64 in the final sample . Volunteers were paid a small honorarium at the end of the study. The study was approved by the UCL Research Ethics Committee, and all participants gave signed informed consent.2.2Participants were randomised to propranolol or placebo conditions stratified by sex to ensure equal numbers of men and women in each group. None of the researchers involved in the study were aware of the treatment condition to which individuals were assigned. Propranolol is a non-selective beta-blocker, inhibiting the effects of catecholamines on both β1- and β2-adrenoceptors. Participants were administered 80 mg of sustained-release propranolol or identical placebo once a day after breakfast for 7 days. The dose was selected as the minimum recommended clinical dose for the treatment of hypertension, and was therefore deemed appropriate for healthy volunteers in order to minimise the likelihood of side effects.2.3Participants attended a brief session in the laboratory at which body composition was measured and a questionnaire containing demographic and psychosocial measures was completed. They received a bottle containing 12 pills of the study medication and were instructed to take one capsule every morning after breakfast for the following 7 days. Participants were advised not to take any other medications or herbal remedies while taking part in the study and to avoid alcohol and vigorous physical activity.Study participants returned 7 days later for the laboratory stress session either in the morning (9:00 h) or afternoon (13:30 h), bringing back the pill bottle so that the remaining capsules could be counted. They were instructed not to exercise before the session, not to drink alcohol the evening before, and not to consume any caffeine on the morning of the testing day. They were asked to eat a light breakfast and/or lunch. Anxiety and positive affect over the past week were assessed. Then a Portapres-2  was fitted for the continuous monitoring of BP and heart rate, and an intravenous cannula was inserted for blood sample collection. The participant subsequently rested quietly for 30 min, followed by the baseline blood draw. Participants also rated subjective stress.A behavioural task battery consisting of three tasks was then administered. First was a socially evaluative public speaking task as previously used by our group . The par2.4Body composition was assessed with a Tanita Body Composition Monitor (BC-418MA) from which body mass index (BMI) was derived. Anxiety over the past week was measured with the 7-item anxiety subscale of the Hospital Anxiety and Depression Scale (HADS) , and posBlood samples were drawn into EDTA tubes and immediately centrifuged at 2500 rpm for 10 min at room temperature. Plasma was removed and aliquoted into 0.5 ml portions and stored at -80° C until analysis. Plasma IL-6 was analysed with Quantikine high sensitivity two-site enzyme-linked immunosorbent assay (ELISA) . The minimum limit of detection was between 0.016 and 0.110 pg/ml. IL-1Ra were analysed in duplicate using fluorescent-labelled capture antibody beads , and assayed using a Bio-Plex 200 Luminex system . The limit of detection for IL-1Ra was 2.3 pg/ml. The mean intra-assay coefficient of variations (CVs) for IL-6 and IL-1Ra were 7.3% and 4.6%, and inter-assay CVs were 7.7% and 6% respectively.2.5Systolic and diastolic BP and heart rate were averaged over the following 5 min periods: 25–30 min of the initial resting period (baseline), the 5 min of each behavioural task trial, min 15–20, 40–45 and min 70–75 following tasks. Differences in cardiovascular activity across task trials did not relate to pharmacological treatment, so the 3 task periods were averaged. Missing data for individual trials meant that 57 were included in analyses of IL-6 analyses, 59 in analyses of IL-1Ra, and 56 for BP and heart rate. Differences between treatment groups in background characteristics were analysed using analysis of variance with sex and treatment as between-person factors. Differences in baseline physiology were explored with analysis of covariance including age, BMI and smoking status as covariates. We analysed subjective stress, inflammatory and cardiovascular responses over the experimental session with repeated measures analysis of variance with sex and treatment as between-person factors, and trial as the within-person factor, again adjusting for age, BMI and smoking. Stress responses were analysed by computing change scores between baseline and task and recovery trials, including baseline levels as covariates. The majority of sessions (61.3%) took place in the morning, but the statistical results were unchanged when time of day was included as an additional covariate. Significant sex by trial interactions were followed by separate analyses of men and women. Statistical analyses were performed using SPSS version 24.0 .3Randomization of the 20 men and 44 women in this study resulted in equally sized propranolol and placebo groups, as detailed in 3.1F = 7.16, p = 0.010). Baseline concentration of IL-1Ra was lower in the propranolol than placebo condition  = 6.71, p = 0.012). There was a main effect of sex in systolic BP  = 4.34, p = 0.042), together with a sex by treatment interaction  = 4.95, p = 0.031). The sex by treatment interaction was significant for diastolic BP as well  = 5.46, p = 0.024). As can be seen in F = 11.73, p < 0.001), with heart rates being substantially lower in the propranolol than placebo groups.Physiological activity at baseline is summarized in 3.2F = 45.61, p < 0.001). There were no differences between treatment conditions or sexes in these responses. Similarly, ratings of task difficulty  did not vary between propranolol and placebo conditions (p = 0.32).Stress ratings increased significantly in response to tasks, from a baseline mean of 1.85 ± 0.96 to 3.80 ± 1.36, 3.82 ± 1.53, and 4.26 ± 1.66 after the speech, mirror tracing, and arithmetic tasks respectively  = 4.67, p = 0.036) and trial  = 36.00, p < 0.001), together with a significant sex by trial interaction  = 3.92, p = 0.010). Men and women were subsequently analysed separately. As can be seen in F = 8.10, p < 0.001), with a marked attenuation of IL-6 response to stress in men taking propranolol compared with placebo. This was confirmed in analyses of differences in IL-6 concentration at 45 min and 75 min after tasks compared with baseline, with smaller increases at both time points in the propranolol than placebo group  = 12.00, p = 0.005 and F = 15.28, p = 0.002 respectively). By contrast, there were no treatment effects among women.Analysis of plasma IL-6 concentrations over the session showed main effects for sex  = 4.55, p = 0.038), but no sex difference or trial effects. The lower levels of IL1-Ra already identified at baseline  = 54.30, p < 0.001), with partial recovery towards baseline over the post-task period. Systolic BP in men remained somewhat lower in the propranolol than placebo condition, but there were no significant differences in BP responses to tasks related to treatment in either men or women. A similar pattern was observed for diastolic BP (results not shown).Systolic BP responses across the session are shown in F = 59.49 and F = 18.25 respectively, both p < 0.001). Heart rate increased during tasks and reverted to baseline levels during the recovery period. The effect of propranolol was more marked in women than men, since there were significant treatment effects in all trials (p < 0.001), whereas differences between treatments were not present in the recovery period in men.The analysis of heart rate showed main effects of trial and treatment condition  and 75 min , but only among men in the placebo condition. There were no associations among men in the propranolol condition, or among women in either treatment group.Associations between IL and 6 and systolic BP responses to stress were evaluated. Interestingly, systolic BP during tasks was correlated with IL-6 increases at 45 min .


The repeatability of this method was confirmed by inter-assay and intra-assay tests with variability ranging from 0.74 to 2.25%. The results have indicated that the qLAMP assay was a simple, rapid, accurate, sensitive, and specific method for detecting N-GPV, especially on field detection.An infectious disease characterized with short bills and protruding tongues has attacked to meat ducks in China since March 2015, which has caused ducks poor growth and enormous economic losses to duck industry of China. It was eventually proved to be caused by parvovirus after pathogen isolation and identification. As the genomic sequence analysis showed, this pathogen shared 90.8–94.6% of nucleotide identity with goose parvovirus (GPV), and it was called duck-origin novel goose parvovirus (N-GPV). In this study, a quantitative loop-mediated isothermal amplification (qLAMP) assay was developed for the rapid diagnosis of N-GPV. A set of four specific primers, two inner and two outer, were designed targeting at VP3 gene, which could be completed within 60 min at 65°C in water bath or on a real-time PCR instrument for quantitative analysis. Specificity test of LAMP assay showed that there was no cross-reactivity between N-GPV and other duck pathogens, and the detection limit of qLAMP assay was 1.0 × 10 Parvoviridae family with a single-stranded DNA genome, and has a small, nonenveloped, icosahedral capsid packaging a single-stranded DNA genome of approximately 5,100 nucleotides. There are two open reading frames (ORFs) encoding non-structural proteins (NS), and capsid proteins (VP)  .Bst DNA polymerase large fragment with high strand displacement activity and a set of specific primers that recognizes six distinct sequences in the target DNA  has attracted intense attention, which relies on auto-cycling strand displacement DNA synthesis by the rget DNA . In addirget DNA , which crget DNA . Resultsrget DNA .® is less inhibitory to PCR and less likely to cause nonspecific amplification  for amplifying VP3 gene of N-GPV was established. Amplification curves and good linear relationship have been obtained. Specificity of this method was determined by GPV and other duck-origin viruses, such as duck plague virus, duck tembusu virus, duck hepatitis virus, duck reovirus, Muscovy duck parvovirus, and H9N2-AIV. In addition, sensitivity of qLAMP was carried out with plasmid substances after ten-fold series dilutions. These test results above demonstrated that this method has advantages with strong specificity, high sensitivity, easy operated, and low cost so that is valuable and suitable for clinical application in basic level production.N-GPV used in this study was isolated from morbid ducks liver in Liaocheng city, Shandong province, China (named SDLC strain). GPV (HN strain), duck plague virus (ZB strain), duck tembusu virus (GL strain), duck hepatitis virus (LC strain), duck reovirus (HB strain), Muscovy duck parvovirus (GX strain), and duck-origin H9N2-AIV (GT strain) were preserved by the Research Institute of Poultry Disease of Shandong Agricultural University. Sixty clinical liver samples and 60 cloacal swabs were collected from dead ducks and clinical healthy ducks respectively in Shandong, Jiangsu, and Henan province of China in different large-scale duck farms.g for 15 min. The supernatant were filtered through a 0.22 μm filter, and the filtrate was inoculated into duck embryo fibroblasts (DEF) cells. The culture supernatant was harvested after 5 days post-inoculated for three passages, and detected by PCR.The liver samples were homogenized in dulbecco’s modified eagle medium (DMEM), frozen and thawed three times, then centrifuged at 5000 ×DNA was extracted by TIANamp Genomic DNA Kit . RNA was extracted by MiniBEST Universal RNA Extraction Kit .Figure 1). The reference waterfowl parvovirus isolates are listed in Table 1.VP3 gene of N-GPV, GPV, and MDPV were aligned by the MegAlign software, and phylogenetic tree was constructed with MEGA version 7.0 using the neighbor-joining method (http://primerexplorer.jp/elamp4.0.0/index.html) and Primer 5.0 software respectively. Primers including two inner (FIP and BIP), two outer (F3 and B3), and PCR primers are shown in Table 2.A set of four primers and PCR primers were designed based on the VP3 gene of N-GPV using an online program , and analyzed results. High fidelity clone was cultured and extracted plasmid with Pureplasmid Mini Kit . Meanwhile, 1.0 × 107 copies/μL–1.0 × 102 copies/μL substances were used for constructing standard curve.Outer primers were used to amplify target DNA, and the amplified PCR products were purified with a gel extraction kit , cloned into the pMD18-T vector  and transformed into 4, 3.5 μL 10 mM dNTP Mix, 1.25 μL 20 × EvaGreen® dye, 1 μL Bst DNA polymerase, 1 μL target DNA, 11.5 μL nuclease-free water, and performed using 7300 Real time PCR System . Mixtures were incubated at 65°C in water bath for 60 min, and terminated by heating at 80°C for 20 min , or carried out on 7300 Real time PCR system followed by procedure: 60 cycles of 64°C 10 s and 65°C 50 s, fluorescence signals were collected at the end of 65°C step, and the cutoff point for Tt  value was determined as 50 by the previous reported method, 1 μL F3 and B3 primers (10 μM of each), 2.5 μL 10 × Isothemal amplification buffer, 1.5 μL 100 mM MgSOed method. Data wa2O, 1 μL forward primer, 1 μL forward primer, 1 μL reverse primer, and 2 μL DNA, and performed using PCR System . PCR conditions were as follows: 95°C for 5 min, 30 cycles of denaturation (95°C for 30 s), annealing (55°C for 30 s), and extension (72°C for 30 s), followed by a final extension at 72°C for 10 min.The PCR assay was conducted in 20 μL reaction system, containing 10 μL 2 × Ex Taq Mix, 6 μL ddH7–100 copies/μL) of standard substance were used as templates for qLAMP amplification to confirm its sensitivity.Ten-fold series dilutions  and the Tt values (R2= 0.997), with a regression line revealing an average intercept of 51.14 and an average slope of –5.2 .Concentration of positive plasmid was 253.50 ng/μL, determined by DS-11 Spectrophotometer , and the copy number was 7.91 × 107–1.0 × 100 copies/μL) of standard substance, the qLAMP method for N-GPV detection has the lowest limit of 102 copies, and no amplification signals were observed in negative control (NC) . In our strategy, the results of qLAMP assay could not only be evaluated by naked eyes at the endpoint with EvaGreen® fluorescent dye in the UV light  and agarose gel electrophoresis , it could also be quantitatively monitored using the real-time PCR system during the reaction.Performed by ten-fold series dilutions . The specificity of qLAMP assay could also be confirmed by green fluorescence in the UV light , and typical ladder pattern seen in the agarose gel electrophoresis . In addition, no positive results were obtained in negative control (NC), which indicates that this qLAMP assay has a good specificity for N-GPV detection.Specificity test of qLAMP method was carried out by seven duck-origin viruses and a goose-origin virus (mentioned in virus and samples). The results showed that our assay could only detect N-GPV and GPV, while no amplification signals were detected in other viruses (Table 3). These results revealed that the qLAMP method has a high reproducibility and excellent stability in detecting N-GPV.In the intra-assay test, the coefficient of variation of the Tt values varied from 0.74 to 1.81%; In the inter-assay test, the coefficient of variation of the Tt values varied from 0.82 to 2.25% (7–1.0 × 100 copies/μL) were used as templates for LAMP and PCR amplification. Results showed that the detection limit of qLAMP assay was 102 copies, while PCR assay was 104 copies, which indicated the sensitivity of LAMP method was 100 times higher than PCR method.Different dilution plasmids  is almost the same as qLAMP assay (1.0 × 102 copies/μL)  , so the Although traditional SYBR Green I-based LAMP assay is a good method with high sensitivity and specificity, adding fluorescence dye after reaction would cause aerosol pollution, thus resulting in a false positive. However, SYBR Green I would inhibit PCR amplification if added before LAMP reaction . So, the2 copies/μL, especially the intra and inter-assay variations, — just 0.74–2.25% only. In addition, the specificity of this method was 100 times higher than traditional PCR assay, and was almost the same as qPCR assay.This study was the first to report the EvaGreen-based LAMP assay for detecting N-GPV. Based on the data above, the detection limit of this method was as low as 1.0 × 10Novel goose parvovirus is newly discovered in recent years, which mainly causes ducks growth retardation and high infection rate to meat ducks, while GPV mainly causes serious death to goslings. According to the view of homology, the N-GPV strain is closer to European GPV and vaccine isolates, while separated from Asian isolates. Therefore, it is clear that N-GPV is a member of GPV-related parvovirus. N-GPV and GPV are almost indistinguishable using this method due to higher nucleotide homology, but it’s also a great method for the detection of N-GPV, because these two pathogens are from different poultry.The qLAMP assay has proved to be a highly sensitive, specific, and reliable diagnostic method that can detect N-GPV in a simple, rapid, and cost-efficient way. It could be undoubtedly used as a point-of-care strategy for clinical laboratories to prevent diseases from breaking out .Conceived and designed the experiments: YD, YT, and JY. Performed the experiments: JY, HC, and ZW. Analyzed the data: JY, XY, and XN. Contributed reagents/materials/analysis tools: JY and HC. Wrote the paper: JY.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bacillus subtilis can confer strong, diverse antipathogenic effects, thereby benefitting the host. Carbon source is an important factor for surfactin production. However, the mechanism that bacteria utilize cellulose, the most abundant substance in the intestines of herbivores, to produce surfactin remains unclear. Here, we used B. subtilis HH2, isolated from the feces of a giant panda, as a model to determine changes in surfactin expression in the presence of different concentrations of cellulose by quantitative polymerase chain reaction and high-performance liquid chromatography. We further investigated the antimicrobial effects of surfactin against three common intestinal pathogens  and its resistance to high temperature (60–121°C), pH (1–12), trypsin , and pepsin . The results showed that the surfactin expressed lowest in bacteria cultured in the presence of 1% glucose medium as the carbon source, whereas increased in an appropriate cellulose concentration (0.67% glucose and 0.33% cellulose). The surfactin could inhibit E. coli and Staphylococcus aureus, but did not affect efficiently for Salmonella enterica. The antibacterial ability of surfactin did not differ according to temperature (60–100°C), pH (2–11), trypsin (100–300 μg/mL), and pepsin , but decreased significantly at extreme environments  compared with that in the control group . In conclusion, our findings indicated that B. subtilis HH2 could increase surfactin expression in an appropriate cellulose environment and thus provide benefits to improve the intestinal health of herbivores.Surfactin secreted by  Bacillus subtilis is a widely used animal intestinal probiotic that can adapt well living in animal intestines and secrete a variety of carbohydrate hydrolase enzymes and antibiotics to facilitate host diet utilization and pathogen inhibition, thereby balancing the gut microbiome [B. subtilis is the biosurfactant surfactin, which confers strong antipathogenic effects and has diverse biological activities [crobiome , 2. One tivities , 4.B. subtilis can utilize a variety of nutrients, including glucose, sucrose, and galactose, for surfactin production [B. subtilis strain HH2 isolated from the feces of a healthy giant panda; this strain showed a good adaptation to the herbivore intestinal cellulose environment and exhibited several probiotic functions based on transcriptional regulation [B. subtilis HH2.Previous studies have illustrated that the bacterial carbon source is an important factor affecting the production of surfactin . Althougoduction , the roloduction . Howevergulation . HoweverB. subtilis HH2 from fresh feces collected from healthy pandas was isolated and identified in our previous study [B. subtilis HH2 was based on partial 16S rRNA gene and gyrA gene sequences for further identification at the subspecies level. The 16S rRNA gene were amplified by polymerase chain reaction (PCR) using 8F (5′-AGAGTTTGATCATGGCTCAG-3′) and 1492R (5′-ACGGTTACCTTGTTACGACTT-3′) primers with the following cycling conditions: 95°C for 5 min; followed by 35 cycles of 95°C for 40 s, 55°C for 45 s, and 72°C for 2 min; and a final extension for 10 min at 72°C [5’-CAGTCAGGAAATGCGTACGTCCTT-3’) and p-gyrA-r (5’-CAAGGTAATGCTCCAGGCATTGCT-3’). Cycling conditions were as follows: 95°C for 5 min; followed by 30 cycles of 95°C for 30 s, 40°C for 1 min, and 72°C for 1 min; and a final extension for 10 min at 72°C [Escherichia coli , Staphylococcus aureus , and Salmonella enterica , were also isolated and identified from the feces of healthy adult giant pandas by 16S rRNA.Permission to conduct the feces sample collection was granted by the director of China Conservation and Research Center for the Giant Panda and the research ethics committee of Sichuan Agricultural University. us study . Phyloge2HPO4, 30 mmol KH2PO4, 25 mmol (NH4)2SO4, 0.5 mmol MgSO4, 10 μmol MnSO4, 22 mg ferric ammonium citrate, 8 g potassium glutamate, 6 g potassium succinate, 1% glucose, 0.5 mmol CaCl2, 5 μmol MnCl2, and 1000 mL ddH2O at pH 7.2. The three other media used in this study included mixed-1, mixed-2, and cellulose media which were formulated in the same manner as the glucose medium, except that the main carbon sources were 0.33% sodium carboxymethylcellulose plus 0.67% glucose (mixed-1), 0.67% sodium carboxymethylcellulose plus 0.33% glucose (mixed-2), or 1% sodium carboxymethylcellulose (cellulose) instead of 1% glucose.Glucose medium was slightly modified from previous studies , 10 and B. subtilis HH2 were extracted using bacterial total DNA/RNA kit  according to the manufacturer’s instruction. Reverse transcription was performed immediately after RNA extraction using RT Reagent Kit with gDNA Eraser .The DNA and RNA of sfp was detected by polymerase chain reaction (PCR) and reverse transcription (RT)-PCR with the primers F (5′-ATGAAGATTTACGGAATTTATATG-3′) and R (5′-TTATAAAAGCTCTTCGTACGAG-3′) [The surfactin gene CGAG-3′) , 11. Cyc600) was equal to approximately 1. After cultivation, the cells were removed by centrifugation at 4°C, 8,000 rpm for 10 min and the resulting supernatant was sterilized again using bacterial filtration (Φ = 0.22 μm). After adjustedpH to 2.0, the cell-free supernatant was centrifuged again at 4°C, 8000 rpm 20 min. The precipitate was then extracted twice with 10 mL methanol, and then adjusted pH to 7.0 with NaOH. Samples were next centrifuged at 10,000 rpm for 5 min, and the collected organic fraction was further processed using a rotary evaporator at 40°C and diluted to 1 mg/mL. The crude extract of surfactin was analyzed by high-performance liquid chromatography  using a C-18 column (250 mm × 4.6 mm) at 25°C. Separation was carried out using a H2O/trifluoroacetate/acetonitrile (ACN) 0.1% (v/v) solvent system monitored at 210 nm. After an initial 2-min wash with 60% ACN, elution was achieved in 60 min at a flow rate of 1 mL/min from 0% to 100% ACN, followedwash 5 min with 100% ACN [Ten microliters of the solution containing the HH2 strain was added to 1 mL LB medium at 37°C for 16 h for activation from refrigerator and then grown in 100 mL LB medium at 37°C in a shaker at 180 rpm for 18 h, at which time the optical density at 600 nm  and specific primers, with the following conditions: 3 min hot start at 95°C followed by 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 40 s. Each sample was run in triplicate. Surfactin expression was quantified using the comparative CT method, and data were analyzed by applying the Relative Expression Software Tool (version REST-384) [The expression of surfactin mRNA was first measured by RT-qPCR using the following primers: srf-F, EST-384) .Surfactin extracted from cells grown in the four media was diluted to 1 mg/mL and then analyzed by HPLC as described above. The areas of the three peaks having the same location as the commercial surfactin solution were calculated to evaluate the peptide moieties.E. coli, Staphylococcus aureus, and Salmonella enterica. We also used reference strains, including E. coli (CCTCC AB 212358), Staphylococcus aureus (CCTCC AB 91053), and Salmonella enterica (CCTCC M 95026), purchased from the China Center for Type Culture Collection (CCTCC). The antimicrobial abilities of surfactin were assessed using the Oxford Cup method [7 CFU/mL with the Oxford Cup. Ten microliters of the HPLC-purified extract was diluted and added at the midpoint of the Oxford Cup. The samples were then cultivated at 37°C for 24 h, and the inhibition zone was measured. Methanol was used as a control. Inhibition zones for each group were measured three times to reduce experimental error.We selected three common gut pathogen species isolated from the giant panda to detect antimicrobial activity, i.e., p method . BrieflyFor analysis of resistance to temperature, pH, and proteases, 0.4 mg/mL surfactin extract samples were processed at 60–121°C in a water bath for 30 min, at pH 1–12 overnight (with the pH adjusted using HCl or NaOH), or with 1–3 mg/mL trypsin (pH = 8) or pepsin  for 4 h. After treatment, the processed surfactin extract was adjusted to the control environment , and antimicrobial activity was then assessed as previously described.P values using t-tests in IBM SPSS statistics software (version 20.0).The expression and antimicrobial effects of surfactin in each group were evaluated three times to reduce error. Significant differences were determined by analysis of B. subtilis HH2 used in the current study were first identified based on 16S rRNA and gyrA genes. The sequencing results are shown in B. subtilis subspecies showed more than 99% homology with our strain, e.g. B. subtilis subsp. subtilis strain SRCM100333, B. subtilis subsp. spizizenii ATCC 6633, according to gyrA gene BLAST results.The strain sfp was also identified by PCR and RT-PCR from the B. subtilis HH2 genome and transcriptome, respectively. Sequencing of the PCR product by BLAST search in the NCBI database showed that the gene had 99% homology with sfp (X63158.1), indicating that this bacterium had the ability to produce surfactin. Next, we used HPLC technology to evaluate surfactin extracts from metabolites of B. subtilis and compared the results with those of commercial surfactin. From this analysis, three peaks of the extract had the same location as the surfactin standard; however, several additional peaks were also observed . However, the antibacterial ability significantly decreased when the temperature reached 121°C (P < 0.05). Additionally, the surfactin extract maintained most of its antibacterial activity within the pH range of 2–11 compared with that at pH 7 (P > 0.05); however, there was a significant decrease in activity at pH 1 and 12 (P < 0.05). In terms of protease resistance, the antimicrobial activity of surfactin extract did not show any significant changes compared with the control group , even at the highest concentration of trypsin or pepsin  in the cellulose group were down-regulated compared with that in the glucose group in order to conserve energy [B. subtilis HH2 showed optimal growth in a glucose medium, accompanied by low surfactin expression. Conversely, the appropriate concentration of cellulose (0.33% cellulose and 0.67% glucose) could increase surfactin expression. Therefore, the growth pressure of cellulose may induce the expression of surfactin. Previous studies have shown that bacterial growth in a stressful environment, e.g., in the presence of fibers, fever, and high salt, can result in selective upregulation of some genes to resist the harsh environmental conditions. The protective effects include the secretion of certain proteins to protect cells [B. subtilis HH2 may increase surfactin expression to inhibit other bacteria and pathogens; thus, this bacterium could yield more nutritional substances and benefit host intestinal health.Generally, glucose culture condition can be beneficial for the expression of most genes and can promote bacterial growth and division. In a previous study, we showed that cellulose was not an ideal carbon source for e energy . Howeverct cells , secretict cells , and secct cells . In an aB. subtilis HH2 showed effective inhibitory activity against E. coli and Staphylococcus aureus, with concentration-dependent effects. One of the most important effect mechanism of the lipopeptide surfactin is leakage and lysis of lipid membranes, which may explain Gram-negative bacteria E. coli was more sensitive than Gram-positive bacteria Staphylococcus aureus [Salmonella enterica. Compared with the commercial standard, our crude extract of surfactin showed some extra peaks and impurities when analyzed by HPLC. Therefore, for more precise calculations, we discarded all additional components and may have also removed some antimicrobial substances and components, thus yielding a different antibacterial spectrum for HH2 compared with standard strains [B. subtilis strains may have divergent antimicrobial abilities. For example, Mireles et al. [B. subtilis has good antibacterial effects against Salmonella enterica, E. coli, and Proteus mirabilis; however, Loiseau et al. [Legionella strains but does not affect any other strains, including Salmonella enterica.In our study, surfactin produced by s aureus . In gene strains . On the s et al.  indicateu et al.  showed tB. subtilis HH2 maintained its antimicrobial activity after being exposed to a range of stresses, including high temperature, extreme pH, and two different proteases. Notably, the surfactin from B. subtilis HH2 showed good stability at high temperatures, even as high as 80–100°C, indicating that this protein may have potential applications for industrial production because food additives may require high temperatures during manufacturing. The stability of surfactin can be attributed to its saddle-shape, which gives it a close-knit structure [Another advantage of surfactin produced by microorganisms is that the protein shows biodegradability under conditions of extreme temperature and pH . In our tructure ; this isB. subtilis HH2 strain enhanced surfactin secretion under conditions of suitable cellulose concentrations, resulting in efficient antimicrobial activity in a variety of environmental conditions. These findings expand our knowledge of surfactin production by herbivore probiotic strains in the presence of cellulose as a carbon source and provide insights into the development of potential panda-specific probiotics. Further studies are required to determine the specific mechanisms mediating the expression of surfactin as a probiotic in an appropriate cellulose environment.In summary, we demonstrated that a panda-derived S1 Table(XLSX)Click here for additional data file.


Spinocerebellar ataxia type 2 (SCA2) is an inherited and still incurable neurodegenerative disorder. Evidence suggests that pro-oxidant agents as well as factors involved in antioxidant cellular defenses are part of SCA2 physiopathology.To assess the influence of superoxide dismutase (SOD3) and catalase (CAT) enzymatic activities on the SCA2 syndrome.Clinical, molecular, and electrophysiological variables, as well as SOD3 and CAT enzymatic activities were evaluated in 97 SCA2 patients and in 64 age- and sex-matched control individuals.ATXN2 gene. SOD3 and CAT enzymatic activities did not show significant associations with the age at onset, severity score, or the studied electrophysiological markers.Spinocerebellar ataxia type 2 patients had significantly lower SOD3 enzymatic activity than the control group. However, there were no differences between patients and controls for CAT enzymatic activity. The effect size for the loss of patients’ SOD3 enzymatic activity was 0.342, corresponding to a moderate effect. SOD3 and CAT enzymatic activities were not associated with the CAG repeat number at the There is a reduced SOD3 enzymatic activity in SCA2 patients with no repercussion on the clinical phenotype. ATXN2 gene. The ATXN2 gene codes for ataxin-2. This is a ubiquitously expressed polyglutamine protein, which has been associated with RNA processing, translational regulation, endocytosis, and cell signaling (Spinocerebellar ataxia type 2 (SCA2) is an inherited neurodegenerative disorder reaching the highest worldwide prevalence rate in Holguín province, Cuba . SCA2 isignaling .ATXN7 gene produced a concomitant increase in ROS levels, aggregation of the disease protein, and cellular toxicity  and catalase (CAT) enzymatic activities have been probed in several models for polyglutamine disorders , 8. Besiin vitro , and higin vitro , highlig⋅O−) to water. First, ⋅O− is converted to hydrogen peroxide (H2O2) by SOD, while in a second step, H2O2 is converted into oxygen and water by CAT  was defined as the onset of motor impairment. Evolution time (ET) was defined as the time elapsed between clinical debut and the time when the last neurologic evaluation was made. Clinical severity was estimated by using the Scale for the assessment and rating of ataxia (SARA) .Horizontal and vertical saccade eye movements were recorded binocularly with silver–silver chloride electrodes over right and left outer canthus and a two-channel Otoscreen AC electronystagmography  with a band-pass filter of 0.02–70 Hz, a sensitivity of 200 μV/division, a time base of 1,000 ms/division, a time constant of 8 s, and a sampling rate of 200 Hz. Saccade eye movements were elicited with a circular white target subtending an angle of 0.7° on a black background. The distance between patient and monitor as well as the head position was controlled by chin/head supports. At least 10 horizontal centrifugal saccades in either direction were recorded for each 10°, 20°, 30°, and 60° predictable amplitudes. Comparison of independent calibrations at a 30° angle before and after all recordings was used to control against artifacts.2O2 substrates were used. One unit of SOD3 enzymatic activity was defined as the amount of SOD3 that inhibits the pyrogallol auto-oxidation to a 50%. Similarly, one unit of CAT enzymatic activity was defined as the amount of CAT that catalyzes the reduction of 1 µmol of hydrogen peroxide per minute.Serum SOD3 and CAT enzymatic activities—expressed as U/L and UI/L, respectively—were measured at 37°C by using standard methods , 16. BufSerum samples were obtained from blood by centrifugation at 3,000 rpm and 4°C during 10 minutes. Samples were freezed at −20°C until used. Every sample was analyzed in triplicate using a Biomate-3 Spectrophotometer .Nucleon II standard protocol . The CAG repeat at the ATXN2 locus was amplified by PCR using UH10/UH13 oligonucleotide primers .Seric SOD3 and CAT enzymatic activities were measured in 161 individuals—66 females and 95 males—aged 15–75 years (mean age: 40.7 years). Neither SOD3 nor CAT enzymatic activities showed a Gaussian distribution  or LogCAT  activities between males and females. In addition, no significant correlations were found between age and LogSOD3 (p = 0.366) or LogCAT (p = 0.275) activities. Log transformed SOD3 and CAT enzymatic activities were highly significantly correlated .SOD3 enzymatic activity varied between 1.16 and 4.78 KU/L, while CAT enzymatic activity was in the range of 3.52–67.61 KUI/L. Mean (SD) SOD3 enzymatic and CAT enzymatic activities were 2.41 (0.80) KU/L and 20.65 (10.85) KUI/L, respectively. There were no significant differences for LogSOD  SOD3 and CAT enzymatic activities were 2.14 (1.45) KU/L and 18.62 (1.48) KUI/L, respectively. On the contrary, among control individuals, the mean (SD) SOD3 and CAT enzymatic activities were 2.40 (1.35) KU/L and 17.78 (1.78) KUI/L, respectively.t = −2.084, p = 0.039)   Figure B. The efp = 0.070). Log transformed SOD3 and CAT enzymatic activities were not significantly influenced by sex in SCA2 patients  = 0.33/0.33, p = 0.964; CAT (KUI/L) = 1.28/1.25, p = 0.555). A similar picture was obtained in the control group  = 0.39/0.38, p = 0.945; CAT (KUI/L) = 1.24/1.27, p = 0.686). No significant correlations were obtained between age and SOD3 or CAT enzymatic activities (p > 0.05).No significant differences were obtained for Log 1/(SOD3/CAT) quotient between patients and controls  or CAT  enzymatic activities, or the Log 1/(SOD3/CAT) quotient .Descriptive statistics for clinical and molecular markers in the study are shown in Table ATXN2 expanded alleles was obtained. However, the CAG repeat number did not show any significant correlation with the log transformed SOD3  or CAT  enzymatic activities, neither with the log transformed 1/(SOD3/CAT) quotient . Also, there were no significant correlations between the AO and the log transformed SOD3  or CAT  enzymatic activities, neither with the log transformed 1/(SOD3/CAT) quotient . The no significant associations between the AO and SOD3 and CAT enzymatic activities were verified by multiple linear regression adjusting for ATXN2 CAG repeat number (p > 0.05). In addition, the ET was not significantly correlated with any of the measured enzymatic activities .As expected, a significant correlation between the AO and the CAG repeat number in p > 0.05). In addition, the ET, log transformed SOD3 and CAT enzymatic activities, and the 1/(SOD3/CAT) quotient, were not significantly correlated with the maximal saccade velocity neither with saccade latency (Table ATXN2 CAG repeat number (p > 0.05).Since slowing saccade ocular movements is a distinctive clinical sign in SCA2, potential associations with SOD3 and CAT enzymatic activities were investigated. Maximal saccade velocity at 10°, 30°, and 60° were significantly correlated with the CAG repeat number. On the contrary, saccade latencies were not significantly correlated with the CAG repeat number , and ATXN2 CAG repeat number  was found. The ATXN2 CAG repeat number was also significantly correlated with the disease’s progression rate (SARA/Log ET) .In a sample of 46 SCA2 patients for which information about SARA score was available, a significant correlation between SARA score and ET , and CAT  enzymatic activities, neither with the 1/(SOD3/CAT) quotient . By multiple linear regression, the lack of significant associations between these clinical variables and the log transformed SOD3 and CAT enzymatic activities and the 1/(SOD3/CAT) quotient was verified, once adjusted for the ET and the ATXN2 CAG repeat number (p > 0.05).However, neither the SARA score nor the progression rate were significantly correlated with the log transformed SOD3 (By measuring biologic markers of oxidative damage, an important role for oxidative stress as part of the molecular physiopathology of neurodegenerative disorders like Alzheimer’s and Huntington’s diseases and FRDA –21 has ba posteriori effect sizes calculations, a much larger sample will be needed to detect any significant effects of CAT enzymatic activity on SCA2.In this study, SCA2 patients showed a moderate decrease in SOD3 activity relative to control individuals, but no difference for CAT activity was noted, suggesting that mild accumulation of superoxide free radical could be taking place in SCA2. The lack of association between SCA2 and CAT enzymatic activity could be due to the lack of statistical power. Actually, based on ATXN2 expanded alleles, suggesting that patients’ SOD3 activity loss is not directly associated with the primary physiopathological events. This result partially agrees with a previous study conducted in SCA2 patients (SOD3 activities in the affected patients were not associated with the CAG repeat number at patients , and simpatients , 25.A possible implication of a decreased SOD3 enzymatic activity in SCA2 patients is an increased susceptibility to oxidative stress. It has been showed that SOD3 null mutant mice develop normally and remain healthy until at least 14 months of age; however, when stress is produced by exposure to >99% oxygen, mutant mice display a considerable reduction in survival time compared to wild-type mice, and an earlier onset of severe lung edema . These fATXN2 CAG repeat number on several clinical markers was showed, explaining about 45% of the age at disease onset, 11% of maximal saccade velocity at 60°, 9% of SARA score, and 16% of the progression rate variability, suggesting the existence of additional factors, genetic and environmental factors, acting as modifiers of the clinical phenotype.In polyglutamine diseases, the CAG repeat expansion mutation is the main genetic factor influencing on clinical variability . In thisOxidative stress has been proposed as a potentially important component of SCA2 physiopathology, leading to the aggravation of the clinical phenotype , 5. PrelBased on the results from this study, it could be suggested that neither SOD3 nor CAT enzymatic activities are potentially useful as biomarkers of SCA2 severity and progression. However, since both SOD3 and CAT enzymatic activities were only measured at the systemic level, further studies will be needed in additional levels—i.e., cerebrospinal fluid—to verify the results and to clarify the role of SOD3 and CAT enzymatic activities in the SCA2 physiopathology. In addition, even when a very large cohort of SCA2 patients was studied, extending this study to presymptomatic individuals will be adding new relevant information regarding the role of these enzymes in early stages of the disease process.There is a reduced SOD3 enzymatic activity in SCA2 patients with no obvious impact on the clinical phenotype.The study protocol was approved by the Research Ethics Committee at the Center for Research and Rehabilitation of Hereditary Ataxias, and a written informed consent was obtained from the affected and control individuals.DA-G, LA-M, and RA-R contributed to the conception and design of the work. DA-G, LA-M, RA-R, RR-L, DC-A, AE-D, LV-P, YG-Z, and YV-M contributed to the acquisition, analysis, and interpretation of data for the work. DA-G and LA-M wrote the paper. DA-G, LA-M, YG-Z, and YV-M revised the paper critically. DA-G, LA-M, RA-R, RR-L, DC-A, AE-D, LV-P, YG-Z, and YV-M approved the final version of the paper to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.