To investigate the clinical efficacy and therapeutic value of posterior decompression reduction, bone grafting fusion, and internal fixation for treatment of symptomatic old thoracolumbar fractures.Retrospective analysis was conducted for 14 patients  with old thoracolumbar fractures who underwent posterior operation. American Spinal Injury Association (ASIA) scores were used to evaluate neurologic function. Vertebral body height, Cobb angle in the sagittal plane, spinal canal volume ratio (%) and bone graft fusion were analyzed by radiography and computed tomography on different follow-up times.Mean follow-up was 27.1 months (23–36 months). Of three patients with ASIA grade A, 2 had improved postoperative urination and defecation, although no classification change. Preoperative ASIA score for eight patients with incomplete injury was grade B; four patients recovered to grade C at final follow-up. Preoperative ASIA score was C in three patients, increased to D in two patients and returned to normal E in one patient. Preoperative results showed average injured vertebra height loss rate decreased from 50.4 to 8.9%; average Cobb angle on the sagittal plane recovered from 39.6 to 6.9°; and the average spinal canal volume ratio recovered from 33.8 to 5.9%. Bony fusion was achieved; local lumbago and leg pain were relieved to some extent. No patients exhibited loosening of the fracture treated by internal fixation, pseudoarthrosis, or other related serious complications.Treatment of old thoracolumbar fractures by posterior decompression reduction, bone grafting fusion, and internal fixation can relieve spinal cord compression, improve neurologic function of some patients (ASIA grades B–C), effectively relieve pain, correct deformity, restore biomechanical stability, and significantly improve quality of life. Thoracolumbar fracture is the most common type of spinal fracture caused by high-energy trauma , with moWith disease progression, thoracolumbar fracture develops into kyphosis, leading to further deterioration of neurologic function. According to the Spine Society of Europe, fractures more than 1 month after injury are considered old fractures . SignifiThis retrospective clinical study assessed 14 patients with severe old thoracolumbar fractures. According to the classification standard revised by the American Society for Orthopedic Surgery (AO) spinal cord injury classification, all cases were anterior and posterior element injuries with distraction (Type B) or rotation (Type C). The purpose of this study was to explore the clinical efficacy and value of posterior decompression reduction, bone grafting fusion, and internal fixation for old thoracolumbar fractures and to observe whether decompression and structural reconstruction of old thoracolumbar fracture can improve the neurologic function and quality of life for patients.From June 2014 to June 2019, 30 patients with old thoracolumbar fractures were identified in this study. A total of 14 patients met the inclusion/exclusion criteria: 9 men and 5 women age 21–61 years, with an average age of 40.1 years. Causes of injury were as follows: high fall for two patients, traffic-related impact for 7, bruising for 3, and coal mine explosions for 2. Damaged segments of the thoracic (T) and lumbar (L) vertebrae were as follows: T10–T11 for four patients, T11–T12 for 4, T12–L1for 2, L1 for 1 and L1–L2 for 3. American Spinal Injury Association (ASIA) classifications of spinal cord injury were as follows: grade A for three patients, grade B for 8, and grade C for 3. The time from injury to surgery was 1.5–6 months, with an average of 3.5 ± 0.9 months. All patients were examined by radiography, computed tomography (CT), and magnetic resonance imaging (MRI) before operation. The kyphotic angle was measured on lateral radiography in terms of angles between the upper surface of the first normal vertebra above the lesion and the lower surface of the first normal vertebra below the lesion , and theInclusion criteria were as follows: (1) radiography, CT, magnetic resonance imaging confirmed old fractures and sagittal Cobb angle ≥ 30°; (2) strict nonsurgical treatment was ineffective for severe low back pain combined with neurologic dysfunction; and (3) fractures and dislocations only involved 2 or fewer vertebrae.Exclusion criteria were as follows: (1) kyphosis caused by tuberculosis or tumor; (2) inability to tolerate surgery because of other serious diseases, such as severe cardiac, hepatic, and renal insufficiency; and (3) blood, immune, and mental health diseases.Under general anesthesia, the patients were placed in a prone position, and vital signs were monitored during the operation. The surgical team chose a posterior median incision to cut the skin, subcutaneous tissue, and deep fascia; exposed the fractured vertebral body and its two upper and lower segments; and exposed the outer edge of the facet joint. In the process of stripping the paravertebral muscle, the pressure of elevator was strictly controlled to prevent further spinal cord injury caused by the fracture of the lamina into the spinal canal.For patients with visible nerve fiber compression, the lamina was retracted with a 2 mm lamina bone bite forceps in the normal lamina space, and the nerve fibers were carefully released to avoid the secondary injury of the spinal cord. The nerve fibers were carefully placed into the dural sac after repair. Pedicle screws were inserted into the upper and lower segments of the injury segment. The spinal cord was carefully pulled with a nerve hook, and the corresponding nerve roots were protected. After reaching the intervertebral space, the upper vertebral cartilage endplate was carefully scraped with a reamer and a scraper to expose the bony endplate.For burst vertebral fractures and anterior vertebral wall, it was essential to pay attention to avoid accidental injury of anterior vertebral vessels. For patients with different pathologic features and degrees of kyphosis, different osteotomy methods were selected for spinal shortening and screw rod system internal fixation. If the reduction of severe fracture was difficult, then the periosteal stripping device was inserted into the bilateral articular process of dislocation space to attempt restoration so that the upper and lower spinous processes are aligned in the sagittal and coronal planes. After a satisfactory reduction was confirmed with the intraoperative C-arm monitor, the pedicle screw could be inserted into the vertebral body under direct vision. Then the SF bracket was placed and pressed to reduce the distance between the two fractured vertebraes and to complete the orthopedic procedure. Interbody spinal fusion with bone particles was trimmed from the spinous process, lamina, and bone fragments of vertebral body. After washing with normal saline, the spinal cord and nerve root were explored again, the herniated spinal cord fibers were retrieved, and the nerve roots were repaired or displaced. The wound was closed layer by layer after placing the drainage tube. The operation duration, intraoperative blood loss, and neuroelectrophysiologic monitoring results were recorded.All patients were routinely treated with antibiotics 24 h after surgery to prevent postoperative infection. The drainage tube was retained for 48–72 h postoperatively, and indwelling catheter was placed. The patients were asked to actively or passively move the limbs to prevent deep vein thrombosis and muscle atrophy. After routine bed rest for 8–12 weeks, patients began to exercise with a walking aid. Patients who could not yet walk choose to perform wheelchair exercise.Patients underwent radiography on postoperative Day 2 to evaluate the degree of decompression and the placement of bone graft and internal fixation. Subsequent follow-up evaluation was at 3, 6, and 12 months postoperatively and annually thereafter. The related complications were observed. Radiography and CT were used to evaluate the recovery of sagittal reduction, decompression, fusion, loss rate of injured vertebral height, Cobb angle correction, and spinal canal occupancy rate. At each follow-up evaluation, radiographic studies obtained from standing and dynamic flexion–extension positions were used to determine fusion status, development or progression of postoperative deformity, and failure of internal fixation. For cases in which the fusion state was indefinite on radiographically, CT scans were performed. Clinical and radiological definition of successful fusion were as follows: no local pain and tenderness, no motor abnormalities, no correction loss, and internal fixation failure. Bone fusion was identified by formation of trabecular bony bridges between contiguous vertebral bodies through CT scans .t test was used to compare the height loss rate of the vertebral body, the Cobb angle on the sagittal plane, the volume ratio of the spinal canal preoperatively, postoperatively, and at the last follow-up. Statistical analysis was conducted using SPSS for Windows , with p < 0.05 considered statistically significant.A paired The same group of chief physicians led the operation of 14 patients with old thoracolumbar fractures. Table Mean operation time was 243 min , and mean bleeding volume was 525 mL .(p < 0.05 vs. preoperative) and to 8.9%  at the final follow-up (p < 0.05 vs. preoperative). The average Cobb angle on the sagittal plane was corrected from 39.6°  preoperatively to 5.7° postoperatively (p < 0.05 vs. preoperative) and 6.9° at the final visit (p < 0.05 vs. preoperative). The average preoperative volume ratio of the spinal canal recovered from 33.8%  to 6.4%  postoperatively (p < 0.05 vs. preoperative) and to 5.9%  at the final follow-up (p < 0.05 vs. preoperative). No significant loss of deformity correction was noted in these patients. Figure All patients showed successful bone fusion, no significant loss of vertebral height and intervertebral height, and a good position of the pedicle screw. The preoperative average height loss decreased from 50.4%  to 7.2%  postoperatively Based on the ASIA grading system, the neurologic deficits for some patients were improved at the final follow-up. No change in ASIA grade was observed for bilateral lower limb paralysis in three patients with grade A, but two patients had improved postoperative urination and defecation. The preoperative ASIA score for eight patients with incomplete injury was grade B, and four patients recovered to grade C at the final follow-up. The preoperative ASIA score was grade C for three patients, which increased to grade D in 2 patients and returned to grade E in 1 patient. Typical patients are shown in Fig. Patients with thoracolumbar fractures, especially those with burst fractures, may experience disease progression to kyphosis if they are not treated promptly. The deformity causes the gravity line of the spine to move forward, which leads to the extension of the resistance arm to maintain the balance and stability of the spine. The anterior and middle columns of the spine bear excessive compressive stress and the columns bear excessive tensile stress. The long-term centrifugal load on the spine aggravates the kyphosis. For these patients who gradually developed into old thoracolumbar fractures, residual fragments in the spinal canal continue to compress nerves or cause spinal instability. In general, spinal deformity, spinal instability, or increased neurologic deficits are surgical indications . PosteriOther studies of thoracolumbar fractures have used different approaches. Machino  used traWhen exposing the surrounding area of the injured vertebra, because of the changes in anatomic structure, especially fracture dislocation, attention should be paid to avoiding damage to important structures such as dura and nerve root.Patients with severe fracture and dislocation often have different degrees of damage to the pedicle; therefore, it is better to insert the pedicel screw using the navigation perspective when operating. In principle, the range of fixation is the upper and lower segments of the injured vertebra, and whether the screw is placed on the injured vertebra depends on the degree of pedicle injury.Attention should be paid to the intensity of surgical traction reduction. First, the ratchet forceps can be used to clamp the lifting reduction, and the reduction or partial reduction can be achieved by combining the horizontal position and axial traction. The traction with excessive force may tear the aorta and inferior vena cava that have been tightly adhered to the anterior vertebral body. If the posterior reduction is difficult, then the periosteal stripping device can be used to place bilateral articular processes of the dislocation vertebral body for poking reset. For old fractures, it is critical to avoid blindly pursuing complete reduction because this approach may damage the aorta and increase the risk of spinal cord injury.For patients with severe kyphosis requiring osteotomy, the articular surface must be excised and the soft tissues of transverse process, lamina, and spinous process outside the facet joint removed. The upper and lower lamina must be completely bitten, grinded to bleeding, and the cancellous bone fragments mixed with artificial bone materials to implant the transverse process space, lamina, and articular process for fusion to ensure the close alignment.For patients with old thoracolumbar fractures, after posterior open reduction, if the bone trabecula and nucleus pulposus structure of the injured vertebral body did not completely reset, then the “empty vertebral body” phenomenon may occur or the surgery may not achieve the purpose of open reduction such that the anterior and middle columns lose the integrity of the structure. If these patients were not given effective bone grafting, internal fixation fatigue, fracture, collapse of fractured vertebral body and loss of correction can occur in the late stage. In this regard, the authors of this current study perform vertebral bone grafting through pedicle at the same time combined with posterior intertransverse process bone grafting to achieve spinal stability. The authors’ surgical purpose is to provide the best conditions for spinal cord recovery.Surgical treatment for severe old thoracolumbar fractures is difficult to perform because the dislocation of the vertebral body results in severe stranded facet joints and because of the presence of hematoma and scar adhesions around the fracture vertebral body, a cauda equina nerve tear, and injury of large blood vessels on the ventral side of the vertebral body . TherefoAnalysis of the statistical results for follow-up of the 14 study cases showed the postoperative sagittal Cobb angle and thoracolumbar sequence improved satisfactorily. During the follow-up period, no further loss of vertebral height and force line occurred, and different degrees of neurologic recovery were observed at final follow-up. In addition, 2 patients with ASIA grade A experienced improvement in urination and defecation, and 4 patients with ASIA grade B advanced to grade C. Among the 3 patients with ASIA grade C, 2 patients were improved to grade D, and 1 patient was restored to grade E. The patients’ back pain was relieved to some extent postoperatively. Previous studies have shown that operation has some complications, such as pseudoarthrosis, epidural hematoma, infection and so on. Urinary tract infection (UTI)occurred in one of 14 patients, he received timely antibiotic treatment. UTI represent a common perioperative complication among elderly patients. For this kind of complication, early detection of symptomatic UTI and targeted antibiotic treatment are very important, perioperative care is also critical to prevent prolonged clinical courses . One patAt present, orthopedic surgeons generally believe that spinal canal decompression can improve neurologic function in patients with thoracolumbar burst fractures . ScholarFor patients with insignificant improvements in postoperative symptoms, however (such as the 3 patients with ASIA grade A and 4 patients with B in this group who did not improve), the necessity of surgery must be analyzed. Some in the field think there is no need for surgery for patients with severe trauma and hopeless neurologic recovery. However, some scholars believe that for patients with old thoracolumbar fractures and spinal cord compression, spinal decompression surgery can improve the local blood supply and metabolism of the spinal cord, which is conducive to nerve rehabilitation, and that surgery can significantly reduce preoperative refractory pain and improve defecation function , even ifIn China, severe thoracolumbar fractures often occur in low-income groups, i.e. injuries caused due to mining work and falls from high altitudes in construction laborers. Often in these patients, the injuries are serious, the clinical recovery is unclear, and high medical expenses are required to be borne. Especially in patients with old fractures, if there is little or no improvement in neurologic function after incurring high medical expenses, further attempts at treatment may cause medical disputes. Therefore, the current authors conducted a preliminary study on whether the surgical treatment of old fractures has a positive effect on the improvement of neurologic function. All the cases collected in this study were severe thoracolumbar fractures, yielding a small number of cases (14 cases) and a short follow-up time (average 27.1 months). The long-term effect of postoperative recovery in these patients is not clear. Although the short-term effect of some patients is ideal, multicenter, large sample, and longer-term follow-up results are still needed to verify the clinical efficacy of this operation.In summary, through this analysis the authors believe that the short-term clinical curative effect for patients with symptomatic old thoracolumbar fractures is meaningful, and the treatment of preoperative injuries is the premise of good curative effect. These results preliminarily confirmed that posterior decompression reduction, bone grafting fusion, and internal fixation for old thoracolumbar fractures can not only relieve pain and correct deformity, but also improve the neurologic function for some patients (ASIA grades B–C), whereas the improvement of neurologic function of patients with ASIA grade A is limited.


Methods: A combined approach using culture techniques and high throughput qPCR were applied to identify ARB and ARGs in rivers along the Lebanese coast. Results: Multidrug-resistant Gram-negative  and Gram-positive bacterial pathogens were isolated. Levels of ARGs were highest in the winter campaign and areas with high anthropogenic activities and population growth with an influx of refugees. Conclusion: Qualitative analysis of ARB and the analysis of the Lebanese estuaries’ resistome revealed critical levels of contamination with pathogenic bacteria and provided significant information about the spread of ARGs in anthropogenically impacted estuaries. Anthropogenic pressure is known to be a key driver of antimicrobial resistance (AMR) dissemination in the environment. Especially in lower income countries, with poor infrastructure, the level of AMR dissemination is high. Therefore, we assessed the levels and diversity of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in Lebanese rivers at estuaries’ sites ( Antimicrobial resistance (AMR) is a One Health crisis aggravated by the lack of water and pollution management on a global scale ,2. AnthrThe surveillance of AMR in the environment is often assessed through ARG quantification ,20. HighEnterobacterales (ESBL-E), multidrug-resistant Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus (MRSA), and ii) the resistome through the detection of ARGs and MGEs.Estuaries are transitional zones between rivers and sea bodies ,25, exhiEnterobacterales and 41 and 43 CFUs resistant Pseudomonas spp. in spring and winter, respectively. However, among the 10,064 CFUs in winter, 104 corresponded to the same bacterial species  in the Beirut estuary. A more diverse panel of Enterobacterales species was isolated in spring. Enterobacterales and/or Pseudomonas spp. strains were isolated from all samples, except the Bared River. Moreover, Enterobacterales were not detected in the Kaleb River and Pseudomonas spp. were not found in the Zahnari river. MRSA isolates were detected only in spring. In total, we isolated 19 different bacterial species in different quantities in spring and winter from the estuarine water samples along the Lebanon coastline  . From 1 Enterobacterales (33) and Pseudomonas (39) strains. The results are shown in Enterobacterales and 6 out of the 39 Pseudomonas strains tested that expressed an ESBL phenotype detected with the double-disk synergy test according to the European Committee on Antimicrobial Susceptibility (EUCAST) v8 guidelines. We performed susceptibility testing for a panel of Pseudomonas in spring were susceptible to gentamicin. In winter, we detected two Pseudomonas aeruginosa strains with an ESBL synergy test, one in Janoubi and one in Beirut. We detected five strains resistant to imipenem in spring, three ESBL-positive Pseudomonas luteola strains (two in Aarqa and one in Beirut), and two Enterobacterales strains (one ESBL Klebsiella oxytoca in Aarqa and one Serratia marcescens in Qadicha). No resistant strains to imipenem were detected in winter.Resistance towards the tested antibiotics was broadly disseminated. All tested strains of Total DNA was extracted from triplicate river samples collected at the 12 estuaries of the major Lebanese rivers. The resistome analysis could only be performed on 10 rivers during both sampling campaigns since for 2 rivers DNA concentration was not sufficient. Overall, a higher normalized abundance of the targeted resistome was detected during the winter sampling campaign . NormaliThe marker IncP-1 was detected in all river estuaries. IncP-1 plasmids are highly promiscuous and considered as anthropogenic markers in the environment. They frequently carry multiple ARGs and are suggested to be important vectors of ARGs and integrons .strB, spc , the mefA gene (conferring resistance to macrolides), the tetracycline resistance gene tetM, and the resistance gene sulA conferring resistance to sulphonamides, were also detected in higher normalized abundance in different rivers during the winter sampling campaign.Noticeably, transposase genes that are associated with the dissemination of ARGs amongst bacteria were detected in high normalized abundance in most river samples, especially during the winter sampling campaign. Individual resistance genes such as the p values corresponding to all the significant variations are available in Variation in normalized abundance for the different gene families in the estuaries during the two sampling campaigns in winter and spring was assessed using the non-parametric Wilcoxon’s test. Although we observed a significant increase for most ARG families in all samples from spring to winter, the mean normalized abundances of tetracyclines and β-Lactams, decreased from spring to winter in the Qadicha and Damour estuaries. In Qadicha, the aminoglycoside, macrolide, tetracycline, multidrug efflux, and transposase genes decreased significantly from spring to winter.Escherichia coli and coliforms, reaching 70.4% of the rivers in the North and 60% in the Bekaa region, which was above the acceptable threshold according to the French SEQ-EAU-2003 recommendations for irrigation water [Here we show that high levels of ARB and ARGs could be detected in all studied Lebanese rivers during all time points. Previous studies underlined the contamination of Lebanese rivers with fecal bacteria, especially on water .In our study, we showed that the levels of ARB and ARGs varied according to the location.Enterobacterales ) in winter and Pseudomonas luteola (104) in spring) were detected in the estuary of the capital Beirut. The highest normalized abundance of ARGs was observed in the Qadicha estuary in spring. The Qadicha river is a small river impacted by population growth, high industrial activities, and a chronic default of wastewater treatment. It has been shown that the estuary of this river was highly contaminated with pollutants [Enterobacterales was isolated from the Aarqa estuary in spring 2017, while a higher bacterial count was observed in winter (Klebsiella pneumoniae 50 CFU). The Aarqa estuary is located on the northern rural coast, inhabited by local and refugee communities with high agricultural activities.The Lebanese coastal belt from Tripoli to Tyre is an urbanized zone with significant agricultural activities ,34. The llutants , which mInterestingly, we observed particularly high levels of ARGs in the North , which cRiver water levels in Lebanon increase during spring due to the rise of underground water and the snowmelt . The difEnterobacterales strains and 6/39 Pseudomonas strains expressing an ESBL phenotype. The targeted resistome analysis of the river water samples detected a panel of ESBL-encoding genes, including a high abundance of ESBL-encoding genes that were specifically detected in the Mediterranean Sea [TEMbla and GESbla in most river samples during both seasons.Most bacteria that were isolated from the river estuaries were highly resistant to β Lactams, with 16/33 nean Sea ,41, suchIS6-group and IS6100 genes were detected in high abundance in the estuaries studied.The high normalized abundance detected for transposase genes in the estuaries in both sampling campaigns might be indicative of an increased dissemination of ARGs among the bacteria in Lebanese estuaries. Transposons in rivers have been suggested to drive the dissemination of ARGs . It was tetQ, tetW, tetM, and tetO) might be the result of antibiotic selective pressure, considering that tetracyclines persist for a long time in the environment and are used frequently in human and veterinary medicine and aquacultural and agricultural practices [The high prevalence of the tetracycline resistance genes  in Lebanon mainly use secondary systems, although their status of operationality is undetermined .Here, we confirmed the estuaries of Lebanon as putative host-spots for the dissemination of ARGs and resistant bacteria into the Mediterranean Sea. Recreational waters such as rivers and beaches have gained increased attention as having a central role in the persistence, dissemination, and emergence of antibiotic resistance . SpecifiLebanon is located in the eastern Mediterranean area along a coastal length of 220 km  and is cFifteen Lebanese rivers spread through or at the extension of the Mount of Lebanon for a dozen of km eastward before discharging in small catchment areas leading to the Mediterranean Sea . The rivn = 72 river water samples: 36 in spring and 36 in winter (3 samples of 60 mL each taken one after the other per river)  . The samStaphylococcus spp., respectively, with and without antibiotics. For Gram-negative enteric bacteria, we used ceftriaxone (2 mg/L), cefepime (4 mg/L), or ertapenem (0.5 mg/L). For Staphylococcus aureus, we used oxacillin (4 mg/L). All media were incubated at 37 °C for 48 h. For Gram-negative bacteria, species identification was performed with API® 20 NE (for Pseudomonas) or API® 20 E  galleries (Biomérieux). For S. aureus, we used three phenotypic tests: catalase, DNAse, and coagulase.The water samples were analyzed by the spread-plate method, culturing a V = 1 mL volume, using sterile rakes on MacConkey and Mannitol Salt agar selective of Gram-negative bacteria and ® discs used were: amoxicillin–clavulanic acid (20 µg–10 µg), cefepim 30 µg, ceftazidim (10 µg), aztreonam (30 µg), gentamicin (10 µg), amikacin (30 µg), piperacillin–tazobactam (30 µg–6 µg), and imipenem (10 µg) for Gram-negative bacteria, and fusidic acid (10 µg), cefoxitin (30 µg), trimethoprim–sulfamethoxazole (1.25–23.75 µg), gentamicin (10 µg), and ciprofloxacin (5 µg) for S. aureus.The susceptibility testing was performed on Mueller–Hinton agar according to the European Committee on Antimicrobial Susceptibility (EUCAST) v8 guidelines. Antibiotic Bio-Rad® (Qiagen) adapted to water samples. All DNA samples were diluted or concentrated to a final concentration of 10 ng/µL for downstream qPCR and 16S rRNA analysis.For molecular analysis, the three water samples per river were filtered , using a filtration ramp  on a sterile 47 mm diameter filter with a pore size of 0.45 µm . Microorganisms were recovered from filters and subject to DNA extraction for downstream analysis using the DNeasy PowerWaterThe Litani and Ibrahim rivers estuaries were excluded from the analysis, as the respective water samples contained insufficient biomass even after an additional 2 L water volume sampling and DNA extraction. ISSW1, ISS1N, IS6100, IS613, IS6 group, Tn3, ISCEc9, tp614), and integron integrase genes  [We targeted 71 ARGs, 6 heavy metal resistance genes, and 3 genes encoding resistance to quaternary ammonium compounds, 9 MGEs , as described previously . A mean To date, this is the first study to provide an accurate assessment and comparison of the targeted resistome in ten estuaries on the Lebanese coast. A combined approach using culture-based techniques and high throughput qPCR for the detection of ARB and ARGs identified Lebanese river estuaries as hotspots for antimicrobial resistance. This study highlights the need to implement regular antimicrobial resistance surveillance and improvement in wastewater management, in addition to the enforcement of regulations and guidelines stringency for sewage sludge or wastewater reuse, as per the new EU 2020/741 legislation of the European parliament.


We evaluated the effectiveness of Rotarix® under conditions of routine use in Mozambican children hospitalized with acute gastroenteritis (AGE). A test negative case-control analysis was performed on data collected during 2017–2019 from children <5 years old, admitted with AGE in seven sentinel hospital sites in Mozambique. Adjusted VE was calculated for ≥1 dose of vaccine vs. zero doses using unconditional logistic regression, where VE = (1 − aOR) × 100%. VE estimates were stratified by age group, AGE severity, malnutrition, and genotype. Among 689 children eligible for analysis, 23.7% were rotavirus positive (cases) and 76.3% were negative (controls). The adjusted VE of ≥1 dose in children aged 6–11 months was 52.0% , and −24.0%  among children aged 12–23 months. Estimated VE was lower in stunted than non-stunted children  vs. 59% ). Rotavirus vaccination appeared moderately effective against rotavirus gastroenteritis hospitalization in young Mozambican children. VE point estimates were lower in older and stunted children, although confidence intervals were wide and overlapped across strata. These findings provide additional evidence for other high-mortality countries considering rotavirus vaccine introduction.Mozambique introduced monovalent rotavirus vaccine (Rotarix Rotavirus is the most important cause of moderate to severe gastroenteritis in children under five years old . In 2019The introduction of rotavirus vaccine in 82 countries contributed to the reduction of rotavirus prevalence among hospitalized paediatric AGE from 38% to 23% between 2008 and 2016 , and simAs a strategy to estimate the burden of rotavirus in Mozambique, two surveillance systems were implemented in the country. Vigilância Nacional de Diarreias—ViNaDia  was launched by the Instituto Nacional de Saúde (INS) in one hospital in 2014 and extended to five others in 2015, all in urban areas ; another®/GSK, Rixensart, Belgium) through the Expanded Program of Immunization, with doses scheduled at 2 and 3 months of age. Following vaccine introduction, RVGE cases reduced from an average of 39% of paediatric hospitalizations to 13%  genotype and 35%  for non-G1P[8] rotavirus genotype . AnalyseThe present analysis describes the “real-world” VE of monovalent rotavirus vaccine in Mozambican children hospitalized with AGE during 2017–2019. In this setting with a high burden of chronic malnutrition, the adjusted estimate of VE for at least one dose of rotavirus vaccine against admission for RVGE was 35%  in children from 6 to 59 months of age. The estimated VE was higher, 52% , in children from 6 to 11 months of age, which, although non-significant, is similar to the point estimates reported in other low-income countries with high child mortality: 52–86% with a median of 58% . Our VE The lower estimates in low-income countries may be due to factors such as greater and earlier exposure to the risk of rotavirus infection, early and co-infection with other enteropathogens , interfeA stratified analysis of VE by age showed a higher protection in children from 6 to 11 months of age ), compared with older children ≥12 months of age ). Lower VE in children ≥12 months were also found in 12 studies conducted in countries with medium and high mortality . Our finThe estimate of VE against more severe rotavirus infection was higher compared with less severe infection, 54% versus −24%, respectively. Although CI were wide and overlapping, this finding suggests that rotavirus vaccine may be most impactful in preventing severe infection in Mozambican children. This result is similar to that reported in nine countries with medium- and high-mortality, where the median VE among children with Vesikari score ≥ 11 points was 54% . Our resIn Mozambique, more than 40% of children less than five years old suffer from chronic malnutrition . MalnutrThe low estimate of overall VE in Mozambique compared with other countries may be related to the fact that Mozambique has the highest prevalence of chronic malnutrition >40%) in children less than 5 years old compared with other African countries (27.0–34.0%), according to the latest community surveys of each country ,38,39,40% in chilRotavirus G1P[8] was the most common circulating genotype during the study period, and this is the principal component of the monovalent vaccine Rotarix. In the present analysis, the VE was similar for G1P[8] 30%) and non-G1P[8] (35%) rotavirus genotypes, which is not concordant with results reported in Kenya in which they found 60% VE against G1P[8] and only 31% for G2P[4] . Other p% and nonThe present analysis had some limitations. A major limitation was a low sample size combined with high vaccination coverage, which limited statistical power, especially in children ≥12 months, and made it difficult to control potential confounders such as markers of socioeconomic status. Achieved statistical power to detect a VE of 50% was <80% for our sample size, and lower for stratified analyses. For this reason, it is not surprising that none of the estimates of VE were statistically significant, and that confidence intervals were wide. We also had to exclude 83 children from the analysis because they did not have confirmed vaccination status. Our inability to include data from immediately following vaccine introduction , due to the inconsistent collection of vaccination cards, was another limitation. However, a strength of this analysis is the breadth of data available, which allowed us to stratify VE estimates by many characteristics of interest, to identify factors that may play a role in the lower VE estimate in the country. Additionally, although estimates were imprecise, the general findings and patterns are in line with previous research, suggesting that our conclusions are reasonable despite the limitations of the data.In conclusion, at least one dose of rotavirus vaccine was estimated to have 52% effectiveness against rotavirus admissions among children aged 6–11 months old in Mozambique, a country with a high burden of chronic malnutrition. The VE estimate from Mozambique is lower than that seen in many other African countries, possibly due to the high burden of chronic malnutrition in children under five years old. This analysis was not powered to detect statistical differences in VE stratified by nutritional status, although stunting appeared to diminish VE. It would be important to understand the role of stunting in vaccine efficacy/effectiveness in Mozambican children using studies with appropriate design and adequate sample size. Despite our inability to detect significant differences, our findings are generally in line with other studies showing the benefits of rotavirus vaccination under conditions of “real-world” usage.


In nature, nothing is wasted, not even waste. Dung, composed of metabolic trash and leftovers of food, is a high‐quality resource and the object of fierce competition. Over 800 dung beetle species (Scarabaeinae) compete in the South African dung habitat and more than 100 species can colonize a single dung pat. To coexist in the same space, using the same food, beetles divide the day between them. However, detailed diel activity periods and associated morphological adaptations have been largely overlooked in these dung‐loving insects. To address this, we used a high‐frequency trapping design to establish the diel activity period of 44 dung beetle species in their South Africa communities. This allowed us to conclude that the dung beetles show a highly refined temporal partitioning strategy, with differences in peak of activity even within the diurnal, crepuscular, and nocturnal guilds, independent of nesting behavior and taxonomic classification. We further analyzed differences in eye and body size of our 44 model species and describe their variability in external eye morphology. In general, nocturnal species are bigger than crepuscular and diurnal species, and as expected, the absolute and relative eye size is greatest in nocturnal species, followed by crepuscular and then diurnal species. A more surprising finding was that corneal structure (smooth or facetted) is influenced by the activity period of the species, appearing flat in the nocturnal species and highly curved in the diurnal species. The role of the canthus—a cuticular structure that partially or completely divides the dung beetle eye into dorsal and ventral parts—remains a mystery, but the large number of species investigated in this study nevertheless allowed us to reject any correlation between its presence and the nesting behavior or time of activity of the beetles. Dung is the site of fierce competition. To coexist in exactly the same space, using precisely the same food, over 40 species of dung beetles are shown in our paper to finely divide the day between them. This is driven by changes in light, which in turn reflect in morphological adaptations among these species which in many cases override their taxonomic heritage. The role of this cuticular structure is not clear, but has been suggested to protect the eye from abrasion in the soil , Pullen nature reserve, Mpumalanga province (25°34′01.6″S 31°10′42.5″E), and near the town of Bela Bela, Limpopo Province (24°46′04.0″S 27°56′37.3″E). All three sampling locations are in the Savanna biome. More specifically, Stonehenge game farm is in the Eastern Kalahari Bushveld Bioregion, Mafikeng Bushveld (SVk1), Pullen nature reserve is in the Lowveld Bioregion, Malelane Mountain Bushveld (SVl11), and the Bela Bela site is in the Central Bushveld Bioregion, Central Sandy Bushveld (SVcb12)  ecologically as diurnal, nocturnal, or crepuscular based on when they reached their activity peak; (2) morphologically based on the external structure of the eye; and (3) behaviorally as rollers, tunnelers, or dwellers based on their known nesting and dung‐handling behaviors   the diel activity period, depicted as the mean of abundance of the species in the different sampling times over a period of 24 h, plotted with ggplot2 version 3.2.0, (2) a SEM of the right eye to depict the eye type, and (3) the habitus .The variability in external eye structure (faceted/smooth cornea and presence/absence of a canthus) of the 44 dung beetle species was studied using light microscopy and scanning electron microscopy (SEM). Individuals used for the scanning electron microscopy were cleaned with a mixture of water and detergent, rinsed in 75% ethanol, air dried, and sputter coated with gold 65 s, 20 mA). For each of the 12 combinations of ecological , morphological (presence/absence of a complete canthus and faceted/smooth cornea), and behavioral (roller/tunneler) features found, we randomly selected a model species for illustration  Figure  was usedTo investigate associations between eye size and diel activity period, taking into account the effect of body size, we also measured the relative eye area of the 12 model species selected as above. An additional species was sought for each of the 12 combinations to increase the strength of our analysis; however, only seven additional species from the total collected were suitable, giving a total of 19 species. To measure the area of the curved eye surface, the heads of eight to ten individuals of each species  were removed and the right eye was covered with a thin layer of transparent nail polish. After about 20 min, the nail polish was peeled off producing a clear impression of the eye surface which was then cut to be mounted flat on a microscope slide. Images of the flattened impression of the eyes were taken with a stereo microscope  and the absolute area was measured using ImageJ ver. 1.50i Rasband, . For thePhia  and body size (44 species) among diurnal, crepuscular, and nocturnal species, generalized linear mixed models (GLMMs) were fitted using the package lme4  and at Pullen nature reserve  . In addi beetles .3.13.1.1Scarabaeus (Scarabaeolus) carniphilus Davis and Deschodt, Kheper lamarcki (Mac Leay) , and the roller Allogymnopleurus splendidus (Bertoloni) from tribe Gymnopleurini, were mainly active at noon (Table Garreta unicolor (Fahraeus) [tribe Gymnopleurini], the tunnelers Afrodrepanus impressicollis (Boheman) and Liatongus militaris (Laporte) [tribe Oniticellini], and Onthophagus cribripennis D'Orbigny and Onthophagus sugillatus Klug reached their peak of activity midafternoon , 12 with a single, well‐defined peak of activity, and 15 with dual peaks of activity (Tables ) Figure , and PacDrepanellus laticollis (Fahraeus) [tribe Oniticellini] and Onthophagus asperulus D'Orbigny were primarily active in the early morning and around noon (Table Scarabaeus rusticus (Boheman), Sisyphus fasciculatus Boheman   and Gymnopleurus aenescens Wiedemann [tribe Gymnopleurini] (Table Drepanocerus Kirbyi Kirby and Euoniticellus intermedius (Reiche) [tribe Oniticellini] were instead active midmorning and midafternoon (Table Scarabaeus (Scarabaeolus) damarensis Janssens and the tunnelers Onthophagus pallidipennis Fahraeus  Figure  reached on Table . Finallys Figure  and Onthon Table .3.1.2Onthophagus variegatus Fabricius . The tunnelers s Figure , Onthophs Figure , Caccobisk Table . The tun] Figure   [tribe Scarabaeini] , Copris cassius Péringuey, and Copris mesacanthus transvaalensis Nguyen‐phung [tribe Coprini] became active at dusk. These five species were mostly active during the first half of the night with a peak of activity between the early night and midnight (Table Scarabaeus (Scarabaeolus) flavicornis (Boheman)  Figure , however) Figure .3.2p = .72) nor time of flight activity . A less obvious trait, at least to the human eye, is the facetted or smooth cornea surface , this structure was found in the vast majority of the nocturnal species  flavicornis as the exception). The 44 species and 8 tribes of dung beetles examined in this study revealed an assortment of these morphological traits, independent of their taxonomic classification at tribe level; no <12 combinations of ecological  morphological (presence/absence of a complete canthus and faceted/smooth cornea) and behavioral (rollers/tunnelers) features  and the canthus is complete  with the exception of the absence of a canthus combined with smooth cornea . In addia Figure . The sams Figure . Six of s Figure . The core Figure .3.2.3smooth, with S. (Sc.) flavicornis as the exception  flavicornis  compared to the crepuscular  and diurnal species , which also differ significantly in size from each other .Among our sample of 44 study species, we found that the nocturnal species are significantly larger  was significantly greater than the eye area of the crepuscular  and the diurnal  species. Absolute eye area was also greater in the crepuscular species than in the diurnal species  was significantly greater, followed by the crepuscular , and then the diurnal  species. Not unexpectedly, relative eye size was also greater in the crepuscular species than in the diurnal species  among diurnal, crepuscular, and nocturnal species, we next defined the eye area of 19 of the species collected and measured. From this analysis, we found that the absolute eye area of the nocturnal species  carniphilus was mostly active at midmorning, while its congeneric S. (Sc.) damarensis reached a clear, second peak of activity midafternoon. Both species were found on Stonehenge farm . For example, m Tables  and 2. Son Table , while Ds Figure  reaches s Figure , as repos Figure , and Onts Figure  have a lK. signatus of the diurnal species examined was able to cross. This narrow time slot presumably offers warm temperatures along with predator evasion due to the rapidly changing light intensity  was the only truly diurnal species within that sample. Again, the lack of information on activity periods left this hypothesis unverified. Our results clearly show that the corneal structure (faceted/smooth) is strongly correlated with the diel activity period of the dung beetle species rather than to taxonomy or nesting behavior.While the eyes of the 27 diurnal species of this study had an obviously faceted cornea Figure , some crn et al. , studyinS. (Sc.) flavicornis has a facetted cornea  are characterized by the presence of a complete ocular canthus, and the Sisyphini and Coprini species by the absence of the same, the presence/absence of this structure does not necessarily provide a stable taxonomic classification within the subfamily Scarabaeinae. In the tribes Onthophagini and Oniticellini, for example, only 5 of the 16 and 4 of the 5 species included in this study, respectively, showed a complete ocular canthus . The giaIt has been suggested that the ocular canthus protects the eyes of dung beetles both from abrasion while digging and from damage resulting from collisions with objects during flight ; Data curation ; Formal analysis ; Methodology ; Visualization ; Writing‐original draft . Marie Dacke: Conceptualization ; Funding acquisition ; Methodology ; Supervision ; Validation ; Writing‐review & editing . Marcus Byrne: Conceptualization ; Data curation ; Methodology ; Supervision ; Validation ; Writing‐review & editing .Table S1–S2Click here for additional data file.


The objective of this study was to evaluate if ex vivo intact CB nerve activity and in vivo whole body ventilatory responses to hypoxia were modified by alterations in succinate metabolism and mitochondrial ROS (mitoROS) generation in the rat. Application of diethyl succinate (DESucc) caused concentration-dependent increases in chemoafferent frequency measuring approximately 10–30% of that induced by severe hypoxia. Inhibition of mitochondrial succinate metabolism by dimethyl malonate (DMM) evoked basal excitation and attenuated the rise in chemoafferent activity in hypoxia. However, approximately 50% of the response to hypoxia was preserved. MitoTEMPO (MitoT) and 10-(6′-plastoquinonyl) decyltriphenylphosphonium (SKQ1)  decreased chemoafferent activity in hypoxia by approximately 20–50%. In awake animals, MitoT and SKQ1 attenuated the rise in respiratory frequency during hypoxia, and SKQ1 also significantly blunted the overall hypoxic ventilatory response (HVR) by approximately 20%. Thus, whilst the data support a role for succinate and mitoROS in CB and whole body O2-sensing in the rat, they are not the sole mediators. Treatment of the CB with mitochondrial selective antioxidants may offer a new approach for treating CB-related cardiovascular–respiratory disorders.Reflex increases in breathing in response to acute hypoxia are dependent on activation of the carotid body (CB)—A specialised peripheral chemoreceptor. Central to CB O In co hypoxia . Upon st hypoxia ,6. These2 affinity in contrast with other cell types [2 falls below a threshold of between 40–60 mmHg, i.e., considerably above the normal value of less than 5 mmHg observed in other cells [2 from normoxic values and before the metabolism or function of other cells starts to be impaired [What remains controversial is the precise mechanism by which the CB senses hypoxia, with many different mechanisms being proposed . One of ll types ,9,10,11.i [i [+ channel closure and chemostimulation [ndufs2 gene do not display an increase in respiratory frequency when subjected to hypoxia [ndufs2 deficient mice do not exhibit rises in either mitoROS or intracellular Ca2+ in response to hypoxia [2) to ubiquinone (Q) at complex I [A key consideration is the link between mitochondrial inhibition and activation of the downstream chemotransduction cascade ,12. Suggi , a fall i [i ,15, and/i [i . A recenmulation ,18. Mice hypoxia . CB type hypoxia ,18. Impoomplex I . It is complex I ,21.2 sensing is the finding that type I cell hypoxic sensitivity can be retained in the presence of rotenone (complex I inhibitor) by feeding electrons directly to cytochrome c [SDHD (encoding succinate dehydrogenase (SDH) subunit D) display the same neurosecretory response to hypoxia as those obtained from wildtype littermates, raising questions about the importance of succinate in hypoxic chemotransduction [2 sensitivity in the rat.Countering the idea that complex I-derived mitoROS are essential for CB Ochrome c . Furthersduction . No studThe aim of the current study was to evaluate if ex vivo CB chemoafferent activity and in vivo ventilatory responses to hypoxia could be modified by alterations in succinate metabolism and mitoROS signalling in the rat.n = 98, 5–10 weeks, 120–390 g) were purchased from Charles River, UK. Animals were housed in individually ventilated cages (n = 2–4 per cage) under standard conditions: 12:12 h light:dark cycle (lights on at 0700), 22 °C and 55% humidity. Food and water were available ad libitum. Animals were killed humanely by either exposure to carbon dioxide gas in a rising concentration or by dislocation of the neck .All procedures were performed in accordance with UK Animals (Scientific Procedures) Act 1986 and approved by the UK Home Office (PPL number PF4C074AD) and by the Animal Welfare and Ethical Review Body (AWERB) at the University of Birmingham. Adult male Wistar rats  as previously described [CBs were isolated from adult male Wistar rats under deep non-recovery terminal inhalation anaesthesia , for 20–30 min.Intact carotid bifurcations containing the carotid sinus nerve (CSN) and CB were removed and animals immediately killed by cervical dislocation. The tissue was transferred to a recording chamber (volume of approximately 0.2 mL) with a Sylgard 184 base  and continuously superfused with a bicarbonate buffered Krebs solution containing, in mM: 119 NaCl, 4.5 KCl, 1.2 NaHecording ,27, the Extracellular action potential recordings of single and few-fibre units were recorded from the cut end of the CSN using borosilicate glass pipettes as described ,29. Acqu2 was continuously measured (100 Hz) using an O2 electrode (ISO2) and O2 meter . Basal activity was measured at ca. 300 mmHg and hypoxic responses were induced by a slow ramp down to ca. 100 mmHg before rapid reversal into hyperoxia . Single fibre frequency was plotted against the superfusate PO2 and data fitted to an exponential curve with offset:y is the discharge frequency (Hz), x is the superfusate PO2 (mmHg), a is the discharge frequency as the PO2 tends to infinity (offset), b is the theoretical frequency when the PO2 is 0 mmHg (minus the offset), and c is the exponential rate constant.Experiments were performed at 37 °C and superfusate POHypoxic responses were performed under control conditions and in the presence of pharmacological agents at concentrations consistent with those shown to modify mitochondrial succinate metabolism and mitoROS generation. These included diethyl Succinate  , hypoxic , or hypercapnic  gas mixture, controlled using Iox2.9.11.8 software . Respiratory flow data were sampled at 1000 Hz and respiratory frequency (Rf), tidal volume (VT), and minute ventilation (VE) were calculated offline.Respiratory parameters in unrestrained awake animals were recorded using whole body plethysmography (WBP) specifically designed for rats as described . Gas flo2, 5 min)/normoxia (5 min) and immediately after to a cycle of hypercapnia /normoxia (10 min). The final 2 min of hypoxic/hypercapnic exposure was used for analysis. Rats were removed from the WBP chamber and received an intraperitoneal (I.P.) injection of either vehicle or mitochondrial antioxidant . All solutions were prepared fresh on the day of experimentation and injection volume did not exceed 1 mL kg−1 bodymass. After one hour in the home cage, rats were placed back into the WBP chamber and the respiratory protocol was repeated.WBP was performed between 8:30 a.m. to 14:00 p.m. Rats were individually placed in the WBP chamber and allowed to acclimatise for 15–30 min. Following acclimatisation, a 5-min baseline was recorded. Rats were then exposed to a cycle of hypoxia  repeated measures one-way analysis of variance (ANOVA) or (iii) repeated measures two-way ANOVA with Tukey or Dunnett’s post hoc analysis where appropriate . Significance was taken as p < 0.05.Data are presented as mean ± SEM or as box-whisker plots with median, mean (shown as +), the box representing the interquartile range and the whiskers extending to outliers. Single points represent individual chemoafferent fibres or animals. Statistical analysis was performed using (i) a paired 2-tailed student’s n = 4. Prolonged exposure of the CB to 5 mM DESucc demonstrated that the chemoafferent frequency did not continue to rise, but rather peaked at 5 min, remained elevated at 15 min before returning to, or slightly below baseline  increased chemoafferent activity in a concentration dependent manner a,b. The baseline c. Even abaseline a. Experibaseline a,b. Thusbaseline c,d. Furtbaseline e–h.2 threshold . In normoxia, DMM caused rapid and reversible chemostimulation in all preparations tested a,b. The  washout c,d.2 threshold for response initiation, suggestive of a decrease in CB O2 sensitivity  did not modify basal Rf, VT, and VE or the ventilatory response to hypoxia and hypercapnia , MitoT had no effect on normoxic Rf but did decrease the rise in Rf induced by hypoxia , the basal Rf was markedly decreased, as was the elevation in Rf caused by hypoxia  and demonstrated consistent breathing patterns and responses to hypoxia and hypercapnia .f in hypoxia and, in the case of SKQ1, the HVR. However, at the level of the CB and in the whole animal, a significant component (50–80%) of the response to hypoxia remains intact. Thus, whilst the data support a role for succinate metabolism and mitoROS being involved in CB and whole body O2 sensing, they are unlikely to be the sole mediators. Interestingly, excessive succinate metabolism causes modest chemoafferent stimulation in normoxia, but blunts activity in hypoxia. The additional inhibitory action of exaggerated succinate and mitoROS generation may have relevance in mediating CB dysfunction in disease.The present study shows that inhibition of succinate metabolism at mitochondrial complex II and mitochondrial antioxidants both partially attenuate the rat CB chemoafferent response to hypoxia. Mitochondrial antioxidants also decrease the rise in R2 sensing in multiple species [m for O2 consistent with the PO2s known to cause type I cell stimulation [2 affinity of CB mitochondria could be due to a unique expression profile of mitochondrial electron transport chain complex subunits [Numerous studies have implicated the CB mitochondria in acute O species ,9,17,35.subunits ,37 or thsubunits .+ channel inhibition, membrane depolarisation, Ca2+ influx, neurotransmitter release, and chemoafferent excitation [ndufs2 prevented hypoxia induced mitochondrial intermembrane ROS generation and rises in intracellular Ca2+ [2 sensing is conserved between species and is present in the rat, albeit to a lesser extent. Validation of a similar role in the human CB is still warranted.There is now much focus on identifying the precise link between mitochondrial inhibition and activation of the downstream chemotransduction cascade: Kcitation . Our datlar Ca2+ ,18. Idenlar Ca2+ ,40,41,42In the presence of DMM, MitoT, and SKQ1, although aspects of the ex vivo CB and in vivo ventilatory response to hypoxia were depressed, they were far from abolished, with around 50–80% being preserved. The stimulation with exogenous succinate led to an increase in the basal nerve discharge frequency, however it did not mimic hypoxia. Excitation by succinate was completely abolished by DMM and was attenuated by MitoT and SKQ1. This is consistent with succinate causing chemostimulation via metabolism at complex II, reverse electron transport (RET), and mitoROS generation. It was hypothesised that co-stimulation of the CB with succinate and hypoxia would potentiate the hypoxic response as reported by Arias-Mayenco and colleagues (2018) in isolated mice type I cells. On the contrary, our data show that such co-stimulation leads to a depression of hypoxic responsiveness in the ex vivo nerve preparation. Therefore, excessive succinate metabolism and mitoROS generation have additional inhibitory actions on CB function that may be relevant in pathophysiology or cellular plasticity e.g., ageing.2 sensing in the rat based on the recent finding that acute lactate administration (up to 20 mM) does not evoke type I cell depolarisation or chemoafferent excitation [Our data support the idea that physiological levels of mitoROS are important in mediating some of the hypoxic chemotransduction, but not all. Changes in MgATP, lactate, and possibly other, as yet unidentified substances, may be necessary for a full response to hypoxia to be evoked ,15. The citation .2S [2S has also been challenged by the findings that mice lacking cystathionine-γ-lyase (CSE) have preserved CB and ventilatory responses to hypoxia [2 stimulus intensity is likely to account for some of these apparent discrepancies, with H2S generation now thought to be more relevant at moderate rather than severe hypoxic intensities [Alternatively, an upregulation of redundant control mechanisms in our experiments could account for the lack of complete abolition of the hypoxic response in the presence of DMM, MitoT, and SKQ1. The pharmacological approach used makes it difficult to separate out potential redundant control mechanisms, with those that are acting in parallel. If redundant mechanisms were at play in the current investigation, then they must have been induced very rapidly as pharmacological interventions were only applied for a maximum of 1 h. The lack of complete elimination of the response to hypoxia in the presence of DMM, MitoT, and SKQ1 also does not rule out the involvement of other mediators independent of mitochondrial function including H2S ,46 and R2S ,48. Whil hypoxia . Again, ensities . TherefoLack of full understanding of the CB chemotransduction cascade undermines any attempts at designing potential treatment options for patients suffering from diseases associated with CB dysfunction, such as heart failure, hypertension, or obstructive sleep apnoea. One of the conclusions that can be drawn from the present study is that different levels of succinate metabolism, and consequently mitoROS, may have opposite effects on the CB activity and the response to hypoxia. The stimulatory impact of the succinate metabolism is likely to be important for initiation of the hypoxic response, whereas when very high excessive levels of succinate metabolism are reached, this may lead to detrimental accumulation of high levels of mitoROS. While ROS are known signalling molecules and are part of many signalling pathways, in severe excess, they cause damage to the cells and individual organelles, such as mitochondria. Providing that one of the hypotheses states that the oxygen sensor is located in the CB mitochondria, a major increase could have a detrimental impact on the overall oxygen sensing mechanism.ROS have also been implicated in CB hyperactivity previously, although the focus so far has been on those derived from angiotensin II and NADPH oxidase ,54,55,56SDHD allele (SDHD+/−) [SDHD gene are one of the causes of pheochromocytoma and paraganglioma, paragangliomas being the most prevalent type of CB cancer [In the current investigation, DMM inhibition of mitochondrial complex II resulted in a pronounced chemoafferent excitation. We speculate that basal stimulation by DMM was a consequence of a slight fall in intracellular MgATP, but this requires validation in future work. Accordingly, previous studies identified persistent type I cell membrane depolarisation in mice lacking one SDHD+/−) . These fB cancer . WhetherA major part of this study was performed on ex vivo nerve preparation rather than isolated type I cells. While chemoafferent recordings are a validated way of assessing the hypoxic sensitivity, it is impossible to determine whether the effects of antioxidants and inhibitors used in this study are limited to type I cells. There could be potential effects on other cell types present in the CB, such as type II cells or the nerve itself and therefore affect the final reading. Similar limitations apply to the in vivo studies as the antioxidants were administered systemically and they were not specifically targeted to the CB mitochondria. Therefore, some systemic effects on the cardiorespiratory system could have altered the response to hypoxia by disrupting ROS signalling. Finally, findings presented here were described in a rodent model, as such further studies are needed to determine whether the same effects are observed in humans.2 sensing both at the level of the intact ex vivo CB and whole body HVR. However, significant proportions of the responses are preserved, suggesting that succinate-mediated mitoROS is not the only relevant signalling pathway. Excessive levels of succinate metabolism impair CB function in hypoxia. Treatment of the CB with mitochondrial selective antioxidants may offer a new approach for treating CB-related cardiovascular and respiratory disorders.Blocking succinate metabolism and the use of mitochondrial antioxidants decreases O


Borrelia burgdorferi, over 30,000 cases are reported in the United States yearly. Patients typically present having early localized disease with fevers, headaches, myalgias, and a single erythema migrans. Usually, oral doxycycline is administered with a good disease prognosis but we report the case of a 58-year-old male who presented with Lyme disease diagnosed by immunoassay; he was treated with doxycycline but was refractory and saw an improvement in his symptoms with IV ceftriaxone.Lyme disease is a vector-borne illness of North America and Europe transmitted by  Lyme disease is a vector-borne illness of North America and Europe transmitted by Borrelia burgdorferi, a spirochete organism carried by Ixodes species of deer ticks . AlthougThe early localized disease is distinguished by the red ring-like expanding rash of erythema migrans at the site of a recent tick bite. Most patients only experience the symptoms of early, localized disease. About 20% of patients develop the early disseminated disease, with the most common symptoms being multiple erythema migrans lesions . Other sThe mainstay of treatment of early Lyme disease, both localized and disseminated, is oral doxycycline. For individuals in whom doxycycline is contraindicated, viable alternatives include amoxicillin and cefuroxime. Complete resolution of all symptoms with any of these three drugs approaches 90% . StudiesWe present a 58-year-old Middle Eastern man with a past medical history of Lyme disease treated for three months with oral doxycycline 100 mg BID in March 2020 diagnosed by strongly positive serology by enzyme linked immunosorbent assays (EIA)  were 0+ on the biceps, triceps, and knees and otherwise 1+. Gait was unable to be assessed due to the patient’s fatigue. His sensations were intact in the extremities. The remainder of the physical exam was unremarkable.His laboratory diagnostics of hematology with differential, chemistry, and urinalysis on admission were unremarkable except for mildly elevated blood glucose of 101 mg/dL and blood eosinophils of 6.1%. His chest X-ray and CT scan on admission were unremarkable. He, however, did present with outpatient MRI that displayed lumbar discogenic disease at the L4-L5 level with nerve root impingement and central stenosis and laboratory elevated IgM and IgG antibodies to Lyme disease.The patient was admitted to the inpatient medical service to be evaluated by infectious disease and neurology who decided to perform a lumbar puncture and peripherally inserted central catheter (PICC) line placement to receive IV ceftriaxone 2 g daily therapy for four weeks. His lumbar puncture displayed a cloudy, pinkish-red sample with high red blood cells at 9660, white blood cells (WBC) high at 13 with 100% neutrophils in differential, the glucose of 68, and total protein high at 85. Routine and fungal cultures of the cerebrospinal fluid (CSF) were negative. He was ultimately discharged on the fourth day of stay after receiving two days of ceftriaxone with subjective improvements in arthralgias and fatigue and displaying 5/5 strength in all extremities, +1 DTRs, and ability to ambulate longer distances without assistance.After discharge, the patient’s HIV1 and 2 antibodies with P24 antigen, rapid plasma reagin (RPR), cryptococcus, angiotensin converting enzyme (ACE) level, complement C3, C4, and CH50, cardiolipin IgA, IgG, IgM antibodies, phospholipid antibodies, anti-dsDNA, and Quantiferon Gold all returned within normal limits. His Lyme antibody was high, greater than 12, and his Lyme Western Blot displayed reactive 18, 28, 30, 39, 41, 58 KG IgG bands with non-reactive IgM bands. His CSF analysis for Borrelia species was negative. Additionally, CSF studies of venereal disease research laboratory (VDRL), IgG, and albumin were all within normal limits. He did display oligoclonal bands of five identical gamma restriction bands indicative of systemic rather than intracerebral synthesis of gamma globulins. His MRI with and without contrast displayed degenerative disc disease of the upper lumbar spine primarily of osteophyte formation without disc herniation or canal stenosis . The pathogenesis of PTLDS is unknown, although elevated levels of IL-23 or CCL19 have been reported . However disease .In this patient, the joint pain and neuropathic symptoms returned after discharge although not as severe as pretreatment. The patient's IV ceftriaxone treatment was stopped after three weeks because he developed a blood clot related to his PICC line and was lost to follow-up. The patient's prior physician treated his joint symptoms and did not recognize that the patient had any neurological Lyme disease, he was told to have neurological symptoms due to discogenic disease. The patient did not have any significant discogenic disease although he had multilevel lumbar foraminal stenosis and nerve root clumping. This patient failed to respond to oral doxycycline for neurological Lyme disease. The patient had several physical findings suggestive of peripheral neuropathy . An electromyography and nerve conduction velocity (EMG-NCV) was never performed because he was lost to follow-up. His lumbar puncture was traumatic and that explains high WBC, but not the WBC differential for high CSF protein.The oligoclonal bands are consistent with systemic inflammation. The MRI suggested arachnoiditis due to nerve root clumping. In Europe, neurological Lyme disease is treated with oral therapy but their strains are different compared to US strains. There are no comparative studies between IV and oral therapy for US strains. This article’s contribution is a warning about the possible lack of effectiveness of oral doxycycline for neurological infections. The newly released national guidelines from the IDSA discussed the treatment issues in detail but the hypothesis about doxycycline efficacy for US treatment is based on in vitro susceptibility testing. The in vitro data does not have the same quality evidence as clinical studies.Fewer than 10% of cases of central nervous system (CNS) Lyme disease will demonstrate predominantly neutrophils in CSF, because this finding is atypical one must consider other diagnoses, such as CNS vasculitis and other connective tissue diseases. An auto-immune evaluation was performed as an outpatient and was not revealing. An outpatient MRI scan of the brain with and without contrast performed within the past six weeks was normal. In the end, we may just conclude that the CSF finding was a reflection of Lyme disease, IV ceftriaxone is entirely appropriate and warranted due to better outcomes.


Amniotic membrane is an effective corneal reconstruction material in veterinary surgery. Cryopreserved amniotic membrane is widely used in practice. Properties of cryopreserved canine amniotic membranes are currently not well studied. This study aimed to compare three properties between canine amniotic membranes cryopreserved for 7 days and 30 days, including tensile strength, transparency, and cell viability. After their respective cryopreservation time, stress–strain curves of the cryopreserved membranes’ tensile strength were assessed using a universal testing machine. Both groups produced J-shaped stress–strain curves with statistically comparable parameters, including maximum stress, strain, and Young’s modulus. The percentage of cell viability was observed by trypan blue staining under a light microscope. Membrane transparency was tested with a spectrophotometer. Transparency tests showed high levels of light transmission and low haze, with no statistical difference between groups. Cell viability was statistically lower in the 30-day cryopreserved group. Tensile strength and transparency of cryopreserved CAM were not significantly impeded for up to 30 days. For CAM to be used as an alternative corneal transplant material in veterinary and regenerative medicine, further research on cell biology, biomechanical properties of the membrane, and cell viability should be conducted. Corneal ulceration is a common ocular disease in veterinary ophthalmology that may lead to numerous complications, including blindness. Surgical treatments are recommended when ulcers have become complicated. Corneal grafting  is the mAmniotic membrane (AM) is sourced from various species: humans , equine Ideally, fresh AM would provide the least degradation of membrane properties and structure ,17. HoweHistological layers of the CAM are comparable to human AM. However, CAM is not attached to the chorion, and it forms blood vessels at a later stage of pregnancy near the umbilical cord . CryopreBiomechanical properties such as tensile strength and transparency of a transplant material are important for corneal reconstruction purposes , and theThe viability of epithelial cells in human AM is significantly lost in most preserved tissue ,33. GlycWhile preserved AM structures of several species have been characterized, there have been no detailed studies of CAM—until now. This study is aimed to investigate and characterize three properties of cryopreserved CAM, namely tensile strength, transparency, and cell viability influenced by storage time for 7 and 30 days.Canine amniotic membranes from healthy puppies (n = 36) were collected from pregnant females with completed vaccination programs. They underwent Caesarean sections at the Small Animal Teaching Hospital, Faculty of Veterinary Science, Chulalongkorn University. Females with systemic inflammation within 3 months, history of abortion or dead fetus, high white blood cell count, or signs of inflammation of CAM were excluded.Washing and storage solutions were freshly prepared in a laminar flow hood with pre-autoclaved equipment. The washing solution was mixed with pre-autoclaved 0.01 M phosphate buffer solution , 1 mL of 5-5-10 mg/mL Penicillin-Streptomycin-Neomycin antibiotic mixture (PSN) , and 0.5 mL of 5 mg/mL Amphotericin B . The storage solution was prepared by mixing 50 mL of pre-autoclaved glycerol , 50 mL of DMEM , 1 mL of PSN, and 0.02 mL of Amphotericin B. The storage solution was stored at 4 °C until use.After a sterile area was prepared, the washing solution and a piece of gauze were laid on a collection tray at the surgical site. Instantly after a removal of a puppy from the uterus, the puppy was separated from the fetal membrane. The membrane was placed on the prepared tray and immediately transported to a laminar flow hood in an insulated box with ice packs. All procedures were completed using aseptic technique.2 for tensile strength test, 4 × 4 cm2 for transparency test, and 2 × 2 cm2 for cell viability test. The cut pieces of nitrocellulose paper were attached to the non-vascularized part of CAM by the stromal side, avoiding the vascularized umbilical cord area. The samples were anatomically located similarly on all CAMs, with the tensile sample located the furthest from the umbilical cord. The CAM samples attached to nitrocellulose membrane were cut and submerged separately in storage solution in a collection vial. All samples were randomly grouped to be cryopreserved at −80 °C for either 7  or 30 days . After storage, samples were thawed at room temperature for 30 min and cleansed with washing solution before tested.In the laminar flow hood, CAM was separated from the fetal membrane and cleansed with the washing solution. Autoclaved nitrocellulose paper was cut to different sizes: 5 × 2 cm2 CAM sample was mounted to a universal testing machine . The nitrocellulose paper was cut across. Tensile strength test was performed automatically by a computerized program , which was set to pull the membrane apart vertically at the rate of 5 mm/min. The pulling force was measured and recorded every 0.05 s until the CAM fractured.The 5 × 2 cmAfter a biological stress–strain curve was generated, values of tensile strength included maximum stress , extensibility, Young’s modulus (MPa), length of toe region, and length of linear region. Young’s modulus constant was calculated from the slope of the stress–strain curve at the linear part by Equation (1).2 CAM sample was carefully stripped away from the nitrocellulose paper. It was then loaded onto the transmission compartment of a spectrophotometer  by using a kraft paper, a clip, and a plastic stand to hold its shape. Quantitative measurement of direct and diffuse transmission of light was automatically performed using total transmittance mode of the software .The 4 × 4 cmThe transparency test results were reported as a percentage of total transmitted light to incident light (%TTRAN) and percentage of diffusely transmitted light to total transmitted light (%Haze); %TTRAN and %Haze were automatically calculated by Equations (2) and (3), respectively.2 CAM sample was stripped away from the nitrocellulose paper, it was mounted on a microscopic slide with the epithelium side up, stained with 20 μL of trypan blue and incubated for 3 min. A sample was visualized under a light microscope. Stained cells represented non-viable cells, while unstained cells represented viable cells. From each CAM sample, microphotographs were randomly taken at 5 fields. The percentage of viable cells compared to all cells (%Viability) was reported as in Equation (4).After the 2 × 2 cmp < 0.05. Since we did not make any assumptions about the parameters, a non-parametric test was selected for this study.All tested parameters, including those of the tensile strength test , transparency test (%TTRAN and %Haze), and cell viability test (%Viability) between 7 d CAM and 30 d CAM, were statistically compared by Mann–Whitney U test  with a significance level of The stress–strain curves of all CAM samples matched the J-shaped stress–strain curve of biological material. Representative stress–strain curves of the two groups are shown in The range of percentage of total transmitted light to incident light (%TTRAN) and diffusely transmitted light to total transmitted light (%Haze) of the 30 d CAM samples was more dispersed compared to the 7 d CAM samples, though more data symmetry was observed . The medp = 0.037).Sheets of uniformly arranged polygonal epithelial cells of CAM were observed . EpithelThis is the first report of the biomechanical properties of CAM. Stress–strain curves generated by CAM matched the J-shaped curve demonstrated in AM of other species, including human , porcineThe biomechanical properties of cryopreserved AM from humans and pigs have been reported ,37. WhenWe found cell viability is poorly preserved in cryopreserved CAM, which is consistent with many previous studies with different measurement methods ,33,41,42Several factors play important roles for clinical selection of membranes for transplantation. Sufficient tissue strength of CAM preserved up to 30 days offers comparable support to corneal damage as the samples preserved for 7 days. Non-significant transparency between the two groups confirms good optical purpose when transplanted. Therefore, we have shown that cryopreservation of CAM between 7 and 30 days resulted in similar properties for both optical and tectonic purposes. Our first investigation of the basic biomechanical properties and cell viability of cryopreserved CAM provides the data to allow future studies to develop this promising material not only for veterinary practice, but also tissue banking. Further study is needed to increase cell viability by focusing on time of tissue storage, storage methods, and media for regenerative therapy. Investigation of longer CAM cryopreservation time is suggested as cryopreserved human AM has been proven to be effective transplant material after being stored for up to 6 months . FurtherConsidering that CAM is an easily available material in a veterinary hospital setting, having information about its basic properties is crucial for creating a more readily available transplantation material to meet its demand at a lower cost, thus improving animal care in veterinary medicine.Tensile strength and transparency are essential properties of CAM, which are required when determining viable corneal transplant materials. These properties of CAM preserved from 7 to 30 days can provide mechanical stability and transparency for the application in veterinary ophthalmology.While the cell viability of CAM is not well preserved by cryopreservation, it is a good processing method for creating a CAM for scaffolding purpose with low graft rejection risk. As for a regenerative medicine purpose, more study is needed to increase cell survival.


Alcyonium acaule, as well as their carbohydrate, protein and lipid contents, was investigated in a warm temperate environment using in-situ observations and biochemical analyses. Polyp activity exhibited a significant variability that was moderately dependent on season, while an aestivation phenomenon in A. acaule  during the warmer months is clearly observed. Carbohydrate concentrations in the coral species showed a significant increase in the late winter and spring seasons, and the lipid content increased during the spring. A higher abundance of lipids and carbohydrates coincided with a higher primary productivity in the water column, as well as with the octocoral reproduction period. In late autumn, there was a depletion of these biomolecules, with protein levels exhibiting great variability across sampling times. Complex alterations driven by climate change could affect the energy fluxes that depend on the dead or alive particles that are intercepted by marine animal forests. The obtained findings show a food shortage in late summer and autumn of the benthic suspension feeder A. acaule through the integrative descriptors of the ecophysiology of these anthozoans. This research contributes to the knowledge of energy storage capabilities in benthic suspension feeders in general, highlighting the importance of understanding the limits of resistance to starvation periods through these indicators.The ecological physiology of anthozoans, as well as their resistance to stressors, are strongly influenced by environmental factors and the availability of resources. The energy budget of anthozoans can vary seasonally in order to find an equilibrium between the available resources and respiration, polyp activity, growth, and reproduction processes. The variation in the biochemical composition of the animal tissues in these organisms results from a combination of the productivity processes of the water column coupled with the reproductive effort and potential starvation periods of the anthozoans. Here, the seasonal variation in the polyp activity of a slow-growing passive suspension feeder, the octocoral  ClimateCorallium rubrum  is colonial with a sessile soft coral thick-bodied and finger-like lobes. This species grows to form a cluster up to 20 cm in height. It is a slow growth organism with a life span of >20 years  belong to the order Alcyonacea, a main structural group of benthic colonial invertebrates found across all latitudes and depths, from shallow waters to the deep sea . Alcyoni20 years . This sonditions . In certnditions .A. acaule in a temperate sea  in relation to temporal coupling with environmental variables. The variation in the biochemical composition of the tissue of the soft coral A. acaule may depend on productivity fluctuations of the water column, and seasonal fluctuations in seston quantity and quality , in Tascons Grossos , ~100 m away from the sampling area , using aAlcyonium acaule over the entire year. Colonies with expanded polyps or completely contracted polyps were observed visually and registered monthly three times a season  by SCUBA diving. Only colonies showing polyps with 100% expansion were assigned as expanded polyps, while colonies with closed polyps were recorded as contracted polyps. Colonies in between these stages were not counted. This alcyonarian also exhibits dormant colonies that were also recorded  were cut and transported in a cooler (4 °C). Samples were frozen in liquid nitrogen within 30–40 min of sampling. The samples were held at −80 °C, freeze-dried at −110 °C and a pressure of 100 mbar, and then stored frozen at −20 °C pending biochemical analyses.−1 OM (Organic Matter). Organic matter was obtained by taking 10–15 mg of the tissue each month (over 1 year), heating the tissue at 80 °C for 48 h and weighing the sample to obtain dry weight. The same tissue was ashed for 4 h at 500 °C. The difference between dry weight and ash weight was considered organic matter  following the protocol of A. acaule among several seasons were assessed across sampling times by multivariate analyses.The differences in (i) polyp expansion and (ii) protein, carbohydrate, lipid concentrations and OM composition of n = 3 for each combination of factors. Multivariate and univariate permutational analysis of variance , post-hoc pairwise tests for the fixed factor were carried out, to ascertain the consistency of the differences among seasons. Because of the restricted number of unique permutations in the pairwise tests, p values were obtained from Monte Carlo samplings. For illustrating differences in the composition of OM, significant terms were plotted using canonical analysis of principal coordinates (CAP)  influenced polyp expansion and organic matter composition of A. acaule, multivariate multiple regression analyses and distance-based Redundancy Analyses  were performed  and Sampling Time (T(S)), as random factor with three levels, nested in Season), with es (CAP)  for the  DistLM, . The ana DistLM, .The tested environmental variables are shown in A. acaule through the entire year is shown in The polyp expansion of A. acaule polyps varied significantly among sampling times within different seasons, while significant seasonal variations had not been observed . In particular, the three considered environmental variables explained cumulatively 20% of polyp expansion variation , followed by the concentrations detected in spring (28.89 ± 2.03 µg mg−1 OM) and summer (24.52 ± 1.59 µg mg−1 OM); the lowest concentrations were reported in autumn with an average of 21.94 ± 1.51 µg mg−1 OM , followed by temperature at −5 m and water transparency, which altogether explained cumulatively 26% of organic matter composition variation  and population density  differently according to species-specific trophic strategies  during the summer period, however the aestivation processes can differ widely among taxa  availability has been discussed . MetabolA. acaule. Polyp activity highlighted a high variability that was only slightly dependent on seasons, while the aestivation phenomenon, a resting period in which the Anthozoan is not capable of any polyp activity, during the warm months has been quantified. This research supports the hypothesis that some Mediterranean benthic suspension feeders exhibit a seasonal pattern of trophic status, characterized by aestivation. Further research will elucidate the complex alterations driven by climate change in marine animal forests and in their processes of carbon immobilization (Our findings demonstrate a food shortage in late summer and late autumn for the benthic suspension feeder lization , as foodlization . The kno10.7717/peerj.12032/supp-1Supplemental Information 1Click here for additional data file.