The World Health Organization recommends regular therapeutic efficacy studies (TES) to monitor the performance of first and second-line anti-malarials. In 2016, efficacy and safety of artemether-lumefantrine (AL) for the treatment of uncomplicated falciparum malaria were assessed through a TES conducted between April and October 2016 at four sentinel sites of Kibaha, Mkuzi, Mlimba, and Ujiji in Tanzania. The study also assessed molecular markers of artemisinin and lumefantrine (partner drug) resistance.Plasmodium falciparum kelch 13 (Pfk13) (codon positions: 440–600) and P. falciparum multi-drug resistance 1 (Pfmdr1) genes , markers of artemisinin and lumefantrine resistance, respectively.Eligible patients were enrolled at the four sites, treated with standard doses of AL, and monitored for 28 days with clinical and laboratory assessments. The main outcomes were PCR corrected cure rates, day 3 positivity rates, safety of AL, and prevalence of single nucleotide polymorphisms in Pfk13 and Pfmdr1 genes, respectively. Six (1.9%) had non-synonymous mutations in Pfk13, none of which had been previously associated with artemisinin resistance. For Pfmdr1, the NFD haplotype  was detected in 134 (39.0%) samples; ranging from 33.0% in Mlimba to 45.5% at Mkuzi. The difference among the four sites was not significant (p = 0.578). All samples had a single copy of the Pfmdr1 gene.Of 344 patients enrolled, three withdrew, six were lost to follow-up; and results were analysed for 335 (97.4%) patients. Two patients had treatment failure (one early treatment failure and one recrudescent infection) after PCR correction, yielding an adequate clinical and parasitological response of > 98%. Day 3 positivity rates ranged from 0 to 5.7%. Common adverse events included cough, abdominal pain, vomiting, and diarrhoea. Two patients had serious adverse events; one died after the first dose of AL and another required hospitalization after the second dose of AL (on day 0) but recovered completely. Of 344 samples collected at enrolment (day 0), 92.7% and 100% were successfully sequenced for Pfk13 mutations, but there was a high prevalence of a Pfmdr1 haplotype associated with reduced sensitivity to lumefantrine (but no reduced efficacy was observed in the subjects). Continued TES and monitoring of markers of resistance to artemisinin and partner drugs is critical for early detection of resistant parasites and to inform evidence-based malaria treatment policies.The study indicated high efficacy of AL and the safety profile was consistent with previous reports. There were no known artemisinin-resistance Trial Registration ClinicalTrials.gov NCT03387631 Despite a decline of malaria burden over the past decade, malaria remains a major public health threat , 2. An ePlasmodium falciparum [Plasmodium falciparum kelch 13 (Pfk13) gene mutations [Artemisinin-based combination therapy ACT is recommended by the World Health Organization (WHO) for the treatment of uncomplicated malaria caused by lciparum . The curlciparum . Tanzanilciparum , and it lciparum . Studieslciparum –12, Kenylciparum , Uganda lciparum , Rwanda lciparum , Burundilciparum , and in lciparum –19 have lciparum  or Plasmutations –23 are rutations , 24, yieP. falciparum genome as markers of resistance to artemisinins and partner drugs of the currently used artemisinin-based combinations. A number of single nucleotide polymorphisms (SNPs) in the Pfk13 gene have been shown to confer resistance to artemisinins [I, N458Y, M476I, Y493H, R539T, I543T, P553L, R561H and C580Y) have been validated as markers of parasite resistance to artemisinins in Southeast Asia [P. falciparum multidrug resistance 1 (Pfmdr1) gene appear to be associated with decreased susceptibility to lumefantrine [Pfmdr1 copy numbers has been suggested to be associated with reduced susceptibility to lumefantrine [Due to the threat of artemisinin drug resistance, WHO recommends regular surveillance  to monitor the performance of anti-malarials in malaria endemic countries. Furthermore, the WHO recommends molecular surveillance based on known polymorphisms (including copy number variations) in the misinins , 26; andast Asia . Polymorfantrine , 29. Genfantrine .In Tanzania, the National Malaria Control Programme (NMCP) and its partners have been implementing therapeutic efficacy studies (TESs) to monitor the efficacy and safety of different anti-malarials , 32, incThis study was carried out at four of the eight NMCP sentinel sites  between April and September 2016. The study sites , about 100 km west of Dar es Salaam . Kibaha has low malaria transmission (< 10%) as reported in previous population surveys –36. MalaMkuzi health centre is located in Muheza district of Tanga region, in northeastern Tanzania. The malaria epidemiological profile of Muheza district has been well characterized and a detailed description given elsewhere , 38. MkuUjiji health centre is located in Kigoma urban district of Kigoma region, on the eastern shores of Lake Tanganyika in the northwestern part of Tanzania. Despite a decline in malaria transmission reported from 2007 onwards –36, 39, Mlimba health centre is located in Kilombero district of Morogoro region and the areas around this site have been extensively studied under the Ifakara Health Institute demographic surveillance system over the past two decades –43. KiloThis was a single-arm prospective in vivo study designed to assess the therapeutic efficacy and safety of AL for the treatment of uncomplicated falciparum malaria and the markers of artemisinin and lumefantrine resistance. Malaria transmission has been decreasing in some of the sites compared to the past and, therefore, thresholds of parasitaemia for low transmission areas recommended by WHO  were used. The age of study participants (6 months to 10 years) was also broadened , 46.Per the WHO protocol, sample size estimates assumed 5% of the enrolled patients would have treatment failure after treatment with AL. At a confidence level of 95% and an estimate precision of 5%, a minimum sample size of 73 patients was required to detect a failure rate ≤ 5% . With a At each of the study sites, potential participants were screened at the outpatient departments using malaria rapid diagnostic tests and microscopy as previously described . PatientTwo blood slides were collected, and one of the slides was stained with 10% Giemsa for 10–15 min and examined by microscopy to detect presence of and an estimated density of malaria parasites. The second blood slide was stained with 3% Giemsa for 30–45 min and used to determine the actual parasite density, species, and presence of gametocytes. Parasitaemia was measured by counting the number of asexual parasites against 200 leucocytes in thick blood films and detection of the different parasite species was done on thin films. Parasite density per µl of blood was calculated by multiplying the total count by 40, assuming that 1 µl of blood had a mean count of 8000 leucocytes . When mo®, Beijing Novartis Pharma Ltd, Beijing China; provided by WHO) for 3 days. Weight-based dosing based on one of three ranges  was done using a fixed dose combination of 20 mg of artemether and 120 mg lumefantrine per tablet, thus patients were given one, two or three tablets from smallest to heaviest, respectively. A full course of AL consisted of 6 doses given twice daily (8 h apart on day 0 and 12-h on days 1 and 2). Patients were observed for 30 min to ensure they did not vomit the study drugs. If vomiting occurred, a repeat dose was given after vomiting stopped. Any patient who persistently vomited the study medication was withdrawn and treated with quinine (injection/intravenous) or artesunate injection according to the national guidelines for management of complicated and severe malaria [Enrolled patients were treated with AL  when patients felt unwell. Parents/guardians were informed and encouraged to bring their children back to the clinic whenever they were unwell without waiting for scheduled visits. Patients who did not show up for their scheduled visits by mid-day were visited at home by a member of the study team and asked to come to the health facility. If a patient had travelled and could not be traced for scheduled follow-up, he/she was classified as lost to follow-up. During the visits, both clinical and parasitological assessments were performed and DBS were also collected. Patients with recurrent infections occurring on day 7 and afterwards were treated with quinine  or artesunate injection based on clinical presentation as per WHO protocol .The safety of AL was monitored by both passive and active methods through interviews with parents/guardian and clinical/laboratory assessments during the 28 days of follow-up. This enabled investigators to capture and record adverse events (AEs) or severe adverse events (SAEs) that occurred after treatment. During scheduled visits, parents/guardians were directly interviewed and asked to report the occurrence, nature, and incidence of any events occurring at home between the follow-up visits. Clinicians took a history, observed patients, and performed a clinical examination during follow-up visits at the study sites. Laboratory tests were appropriately requested and done to determine and capture AEs/SAEs. The reported/captured events were recorded in respective case report forms for each follow-up visit. Any SAE occurring during the study was reported by the principal investigator to the sponsor , NMCP, and the Tanzanian Medical Research Coordinating Committee (MRCC) of NIMR  within 24 h of its occurrence. Reporting of SAEs was done regardless of whether the principal investigator considered the events to be related to the investigated drug or not. Patients with AEs or SAEs were thoroughly assessed and managed accordingly, and the events were also assessed to determine their association with the study drugs. According to the WHO protocol , an AE wPfk13 propeller domain (codon positions: 440–600) and Pfmdr1  were analysed for SNPs. SNPs were called only if they fit the following criteria: (i) they were found on both the forward and reverse reads, (ii) they had a p - value of < 0.0001 , and (iii) they had a minimum strand bias p-value of < 0.0005 when exceeding 65% strand bias, as some errors from sequencing machines are more likely to happen on nearby upstream bases. Mixed-infection and/or heterozygous calls were excluded from the analysis. The 3D7 Pfk13 and Pfmdr1 were used as reference sequences. Detection of Pfmdr1 copy number variants was performed using an Agilent Mx3005 real-time PCR machine  according to previously described protocols [Molecular analysis was performed on all samples collected upon enrolment (day 0) and during follow up in the case of treatment failure. Parasite genomic DNA was extracted from DBS using QIAamp blood mini-kits  according to the manufacturer’s instructions. Molecular markers of anti-malarial drug resistance and microsatellite markers were analysed at the Centers for Disease Control and Prevention (CDC) Malaria Laboratory in Atlanta, USA. Sanger sequences generated in this study were analysed using the Geneious software package . Raw sequence reads were cleaned using default settings, and reads with high-quality scores  below 70% were discarded from further analysis. The rotocols , 48.® V2.6.3  [Samples from 65 patients with recurrent infections were analysed to determine genetic diversity in the study populations using six neutral microsatellite markers  by nested PCR for all except C2M34-313 and C2M69-383 . Fragment size was measured by capillary electrophoresis on ABI 3033 (Applied Biosystems) and scored using GeneMarkerPA, USA) ). PairedPA, USA) . A recruPfmdr1 and Pfk13 genes. Treatment outcomes were classified as either early treatment failure (ETF), late clinical failure (LCF), late parasitological failure (LPF), or adequate clinical and parasitological response (ACPR) before and after PCR correction; based on per protocol method and Kaplan–Meier analysis.The primary end point was PCR corrected parasitological cure on day 28 as per WHO protocol , while sEthical clearance was obtained from MRCC of NIMR, while permission to conduct the study at the health facilities was sought in writing from the relevant regional and district medical authorities. Ethical clearance form CDC was not required because the assessments done at the CDC Malaria Laboratory, using samples without linked identifiers, were determined by the CDC Center of Global Health’s Human Research Protection Coordinator to not constitute engagement in human subjects’ research. Detailed information of the study and benefits as well as its risks were explained to each study participant. Oral and written informed consent were obtained from parents or guardians of all patients before they were screened for possible inclusion into the study. The study was retrospectively registered at ClinicalTrials.gov, No. NCT03387631 (on 2nd January 2018) because of problems within the system which did not allow to register it before the study was launched.10 transformed parasite density at enrolment and age, among the four sites were compared using t test or analysis of variance—ANOVA  or Mann–Whitney U/Kruskal–Wallis test . The prevalence of different haplotypes or SNPs in the Pfmrd1 and Pfk13 genes as well as Pfmdr1 copy numbers were reported. For the Pfmrd1 gene, the analysis focused on the three SNPs  and their corresponding haplotypes (particularly NFD), which have been associated with reduced susceptibility to lumefantrine [YYY) to resistant haplotype (NFD) on day zero and day of recurrence with adjustment for age of patients and the study site . For all statistical tests and comparisons, a p-value < 0.05 (two tailed) was considered to be significant.Data were entered into a Microsoft Access database at the study sites followed by a second entry which was done centrally at NIMR Tanga Centre after the end of data collection. The data were later validated, cleaned, and analysed using STATA for Windows version 11 . Descriptive statistics such as percentages, mean, median, standard deviation, and range were reported as appropriate. In order to automatically generate the treatment outcomes , the data were appropriately formatted and transferred to the WHO Excel software template . Baselinfantrine , 29. LogA total of 963 children were screened between April and October 2016. All sites recruited 88 children except Kibaha, which enrolled 80, making a total of 344 enrolled at the four sites  were lost to follow-up and three (0.9%) withdrew, leaving 335 (97.4%) patients that reached the study end points who were used in per protocol analysis  had mutations in the Pfk13 gene and only 6 (1.9%) samples had non-synonymous mutations, of which none were similar to previously reported SNPs associated with artemisinin resistance. The six Pfk13 non-synonymous mutations included one patient from Mkuzi (R471S), two at Mlimba (A578S and E433D), and three at Ujiji (one with I416V and two with Q613E). All patients with parasitaemia on day 3 post-treatment had infections with Pfk13 wild-type at enrolment. For Pfmdr1, codons N86Y, Y184F and D1246Y were analysed and the prevalence of the different SNPs are presented in Table F mutant was present between 34.1% (Mlimba) and 46.6% (Mkuzi) in all four sites  samples, and its prevalence ranged from 33.0% in Mlimba to 45.5% at Mkuzi, but there were no significant differences (p = 0.578) among the four sites  during recurrent infections. Although patients with the NFD haplotype at baseline had more recurrent infections, the difference was not significant, even after adjusting for age and study site . Both patients with PCR-corrected treat failure (one with ETF and the second with a recrudescent infection) had NFD haplotype at enrolment. The overall median day of recurrent infection was 21 days  and this was similar among the four sites. Recurrent infections among individuals with NFD haplotypes at enrolment had a median of 23 days compared to 21 days for NYD, but the difference was not statistically significant (p = 0.417). Analysis of Pfmdr1 copy number variants was done on all 65 individuals with recurrent infections; all samples had a single copy of the gene.Out of 344 samples collected at enrolment (on day 0), 92.7% (n = 319) and 100% (n = 344) were successfully sequenced for This study reports high efficacy of AL (PCR corrected ACPR > 98%), suggesting that the ACT has retained its ability to treat uncomplicated malaria despite its use in Mainland Tanzania for more than 10 years. Previous studies conducted at these and other sites in Tanzania reported similarly high efficacies of AL . HoweverThis study also showed that AL had a safety profile comparable to previous studies and was well tolerated with minimal AEs. Most of the AEs were minor and mainly reported at the two sites of Mkuzi and Ujiji. Studies conducted in Tanzania , 12 and Two patients had serious adverse events on the day of presentation (day 0), with one child from Mkuzi dying after the first dose of AL. The cause of death was not established and its possible association with AL treatment could not be ascertained. A similar incident of death was reported in a TES which was conducted at Nagaga in 2012 [Pfk13, a molecular marker of artemisinin resistance [Pfk13 non-synonymous mutations in this study was higher than in a previous study which reported a prevalence of 1.2% [Pfk13 at enrolment. These findings do not suggest that artemisinin resistance has emerged in Tanzania [Pfk13 mutations might be increasingly accumulating in Tanzanian parasite populations and thus continued surveillance will be required to monitor the trends of Pfk13 polymorphisms and their possible association with reduced efficacy of artemisinins.Analysis of sistance , reveale of 1.2% . All patTanzania –23 and oTanzania . HoweverPfmdr1 gene showed that 39.0% of the parasites tested had the N86/Y184F/D1246 (NFD) haplotype, a combination associated with reduced susceptibility to lumefantrine in some studies [Pfmdr1 SNPs had higher prevalence than what was reported by previous studies in Mainland Tanzania [Analysis of the  studies . This coTanzania  and ZanzTanzania ; howeverPfmdr1 copy number with reduced susceptibility to lumefantrine [Pfmdr1 gene. This could be related to the fact that mefloquine has never been used as a routine first-line therapy in the study areas.Although studies have associated elevated fantrine , 47, 57,Pfk13 mutations or amplification of the Pfmdr1 gene were identified. A high prevalence of a Pfmdr1 haplotype was detected but corresponding subjects with treatment failure were not found. Continued TESs and monitoring of markers of resistance to artemisinin and partner drugs is critical and should be sustained to facilitate early detection of resistant parasites and to inform evidence-based malaria treatment policies.The findings of this study showed that the efficacy of AL remains high in Tanzania despite the use of this combination for more than 10 years. The safety of AL was consistent with previous reports and no known artemisinin-resistance


An initial screen of the entire mtDNA genome of 6 DR patients detected a higher prevalence of mutations in amplicon P, covering nucleotides 14,443 to 1066 and spanning the control region. Further analysis of 42 subjects showed the presence of putative mutations in amplicon P in 36% (14/39) of DR subjects and in 10% (2/20) non-DR subjects. The prevalence of mutations in DR was not related to the severity of the disease. The detection of a high-prevalence of putative mtDNA mutations within a specific region of the mitochondrial genome supports the view that mtDNA damage contributes to DR. The exact location and functional impact of these mutations remains to be determined.Diabetic retinopathy (DR) is a common complication of diabetes and a major cause of acquired blindness in adults. Mitochondria are cellular organelles involved in energy production which contain mitochondrial DNA (mtDNA). We previously showed that levels of circulating mtDNA were dysregulated in DR patients, and there was some evidence of mtDNA damage. In the current project, our aim was to confirm the presence of, and determine the location and prevalence of, mtDNA mutation in DR. DNA isolated from peripheral blood from diabetes patients ( Diabetic retinopathy (DR), one of the most common complications of diabetes, is the leading cause of visual impairment and blindness in the working age population . DespiteDR is asymptomatic in the initial years of diabetes, during which time molecular changes, currently undetectable in humans, precede visible microvascular changes. Such changes propagate with increased duration of diabetes, however, currently it is not possible to detect them before retinal damage. Screening programs require high-cost imaging tools to assess sight-threatening DR  but are Hyperglycaemia is a key contributor of damage to the eye, leading to increased intracellular reactive oxygen species (ROS), oxidative stress, and damage to DNA, proteins, and lipids . MitochoIn experimental models of DR (cultured cells and rodents), it has been shown that mtDNA damage precedes retinal damage ,8,9 howeWe previously proposed the hypothesis that early changes in mtDNA levels reflect subsequent mitochondrial dysfunction and mtDNA damage . We postIn the current study, we wanted to confirm and extend our earlier finding of mtDNA damage in DR patients . Our aimThis cross-sectional study was carried out using blood samples from 59 individuals . SpecifiIn order to determine potential hotspots of mtDNA damage, we first carried out a pilot study using DNA from 6 DR patients. Total DNA isolated from whole peripheral blood was used to amplify 6 regions of overlapping regions of mtDNA , shown in DNA samples from the entire cohort of 59 patients were used to amplify amplicon P and the overlapping amplicon H’. Mutations in amplicon P were only called if they could be replicated in independent experiments and also confirmed in the overlapping amplicon H’ giving the expected sized bands. An example of the detection of mutations in 3 patients is shown in Although we had predicted mtDNA damage would occur randomly, we noted potential mutational hotspots, i.e., cleaved products of similar sizes in different patients, which we identified by matching bands across patients in relation to ladder and location of undigested band. Sixteen out of the 59 patients showed the presence of mtDNA mutations in amplicon P . AnalysiThe DR group was subdivided as having mild DR (DR-m) or severe DR (DR-s), as we have previously described . The twoWe have previously shown that circulating mtDNA content was dysregulated in patients with DR, showing a biphasic change. Additionally, we reported putative mtDNA damage in 8 DR patients using the “elongase” method , which cInterestingly, a vast majority of studies evaluating mtDNA damage in diabetic complications have identified mtDNA damage by using the amplification of a long-fragment relative to a short-fragment in the mitochondrial DNA ,17,18. TAmplicon P contains a number of genes encoding protein subunits of the electron transport chain, tRNAs and an rRNA molecule. The NADH dehydrogenase 6 (MT-ND6) gene encodes a subunit of complex I, a significant part of the electron transport chain in the inner mitochondrial membrane . Mutatioin-vivo and in-vitro [Diabetic retinopathy, a common complication of diabetes, is not generally regarded as a mitochondrial disease. Traditionally, mitochondrial genetic diseases arise due to mutations in mtDNA itself or in nuclear genes required for mitochondrial function. These diseases are rare, affecting approximately 1 in 4300 adults, and can affect multiple cell types and organ systems leading to symptoms with a range of severity and onset at any age . Howeverin-vitro ,35,36.in-vitro studies that there is an early maladaptive increase in mtDNA which precedes mtDNA damage and bioenergetic deficit [The possibility that mtDNA damage is involved in diabetic complications has been proposed by us previously in work that lead directly to the current study ,12,16, w deficit . Similar deficit ,37. Our In relation to DR, mtDNA damage was detected using the elongase method in a relatively small cross-sectional study involving 11 nondiabetic healthy controls and 11 diabetic individuals, of which 6 were diagnosed with DR, 3 of which had additional microvascular complications such as neuropathy and nephropathy . InteresThe surveyor nuclease method was used in this study for qualitative analyses of mtDNA mutations, i.e., we can report on the presence/absence of potential mutations, but we cannot determine the precise locations of these mutations or the level of heteroplasmy at which they may be present. Next generation sequencing could be useful for verifying our findings and this will enable us to better classify the nature of these mutations. Furthermore, whilst this study features a larger cohort size than many others in the literature, we have not been able to stringently evaluate the relationship between mtDNA damage and diabetes type, the presence of additional microvascular complications, or the clinical progression of DR. In addition, healthy controls without diabetes were not available at the time of this study.In summary, we show evidence of increased prevalence of mtDNA damage in circulation in DR patients. We have identified potential mtDNA mutations in a region spanning genes encoding ND6, cytochrome B, 12S ribosomal RNA, a number of tRNAs, and the noncoding region of the mitochondrial genome. The functional impact of such mutations is not yet known. Mutations were more common in those with DR versus those without. Therefore, our data suggests that mtDNA mutation is associated with DR, however, the nature of this association is yet to be fully elucidated. Damaged out of place mtDNA may directly contribute to increased inflammation in DR-s as it resembles bacterial DNA. If damaged mtDNA contributes to the progression of retinopathy, then the design of strategies for the removal of damaged circulating mtDNA could provide a novel therapeutic target for the treatment and prevention of diabetic retinopathy progression.Patients were recruited from King’s College Hospital  and Guy’s and St Thomas’s hospital clinics   with written informed consent in accordance with the Declaration of Helsinki. Categories for DR severity were defined using the Early Treatment Diabetic Retinopathy Study (ETDRS)  severity2), diastolic blood pressure, and systolic blood pressure (mmHg) were recorded during the visit to the clinic at the time of blood sampling.Metabolic status (HbA1c) and lipid levels were assessed by the hospital clinical lab services using standardized assays. The Jaffe assay was used to measure creatinine. Glomerular Filtration Rate (GFR) was assessed using the Modification of Diet in Renal Disease (MDRD) formula . Body maWhole blood/buffy coat was obtained and frozen within 2 h of collection at −20 °C and transferred to −80 °C within 24 h. Total genomic DNA was isolated from 100 µL thawed sample using the DNeasy Blood and Tissue Kit  and treated with sonication as described in . MtDNA cPotential mutations in mtDNA were detected using the surveyor nuclease method . First, Nuclease digestion of these amplicons was carried out using the Surveyor Nuclease Mutation Detection Kit . A mastermix containing Optimase Polymerase Buffer (6 µL), MgCl2 (1.5 µL), Surveyor enhancer (1 µL), Surveyor Nuclease (1 µL), and ddH2O (35.5 µL) was prepared on ice. For each amplicon, ‘digested’ and ‘undigested’ reactions were prepared by adding 15-µL PCR product to 45-µL mastermix (digested) or 51-µL ddH2O (undigested). Digested reaction mixtures were incubated at 42 °C for 35 min while undigested samples were left at 20 °C. A total of 6 µL of stop solution was added to the digestion mixture to stop the nuclease activity. Products were visualised on 1.5% agarose gel containing ethidium bromide at 80 mV for 40 min.U test was used to compare DR groups after dataset normality was assessed by Shapiro–Wilk’s W test .Statistical analyses of cohort characteristics were performed using GraphPad prism  and IBM SPSS . Nonparametric Mann–Whitney We have shown increased prevalence of mutations in a specific region of the mitochondrial genome in blood samples from patients with DR, and a lower prevalence in diabetes patients without DR. Our data suggests that there may be hotspots of mtDNA damage. The exact nature of these mutations needs to be established in order to determine their potential functional impact. Our data add to the growing body of evidence suggesting that mtDNA is affected in diabetic complications and that acquired hyperglycaemia-induced mtDNA damage may be involved in disease progression. Longitudinal studies could be undertaken to determine if mtDNA is damaged as a consequence of diabetes and whether specific regions of damage contribute to the pathogenesis of DR.


Panthera leo, IUCN Red List Vulnerable) exert top-down pressure on Grevy’s zebra . We examined effects of lion predation on Plain’s zebra  and Grevy’s zebra populations in a 2,105 km2 area defined by lion movements. We used line transect surveys to estimate density of Grevy’s (0.71/km2) and Plain’s (15.9/km2) zebras, and satellite telemetry to measure movements for lions and both zebras. We tracked lions to potential feeding sites to estimate predation rates on zebras. We compared field-based estimates of predation rates on both zebras to random gas models of encounters that result in predation to ask if lions prey preferentially on Grevy’s zebra at a sufficient rate to drive population declines. Lions preyed on Grevy’s zebra significantly less than expected in 15 of 16 (94%) scenarios considered and lions preyed on Plain’s zebras as expected or significantly less than expected in 15 of 16 scenarios. Population trend of Grevy’s zebra indicates that the Kenya population may be stabilizing. Recruitment rate to the population has tripled since 2004, making it unlikely that lions are having an impact on Grevy’s zebras. In Laikipia County, competitive displacement by livestock (Livestock: Grevy’s zebra ratio = 864:1) and interference competition for grass with Plain’s zebra (Plain’s zebra:Grevy’s zebra ratio = 22:1) are most likely the predominant threats to Grevy’s Zebra recovery.When predators are removed or suppressed for generations, prey populations tend to increase and when predators are re-introduced, prey densities should fall back to pre-control levels. In cases of apparent competition where there are alternate abundant and rare prey species, rare species may decline further than expected or disappear altogether. Recently, concern about the impact of recovering predator populations on wildlife in Laikipia County, Kenya, has led to questions of whether lions ( Large carnivores are known to influence the dynamics, distribution and behavior of ungulate prey populations , 2, 3. TUrocyon littoralis) declined after the islands were colonized by golden eagles (Aquila chrysaetos) in response to local extinction of native bald eagles  from DDT [Puma concolor) that prey on endangered Sierra bighorn sheep (Ovis canadensis sierrae) have been selectively removed as part of the Sierra Nevada Bighorn Sheep Recovery Program [Enhydra lutris) and Steller sea lion (Eumetopias jubatus) populations in the Aleutian Islands have been attributed to increased predation by killer whales (Orcinus orca), a result of prey-switching by killer whales after losing their former prey base of great whales to post World War II industrial whaling in the North Pacific [Restoration or maintenance of top predators may conflict with other conservation priorities when successful carnivore conservation in turn threatens a rare prey species. Policy makers and managers then need to address conflicting outcomes in endangered species interactions. In predator-prey relationships, conservation policy and management decisions should be based upon sound data and a thorough understanding of the predator-prey dynamics. In reality, such decisions often are complicated by sociopolitical considerations when predator, prey or both are endangered, protected, charismatic or otherwise in the public eye. For example, off the California coast, endemic Channel Islands foxes  is classified as Endangered on the IUCN Red List, due to a population decline throughout its range from an estimated 13,700 in 1977 to an estimated 2,680 individuals in 2016 throughout the range [Panthera leo), the primary natural predators of Grevy’s zebra, also have been in steep decline, extirpated from over 80% of their historical range and reduced to an estimated population of 20,000–30,000 across Africa [http://ecos.fws.gov/).The Grevy’s zebra ..Equus grs Africa . Lions a2 [2 in the late 1960’s [Laikipia County, Kenya provides refuge for an estimated 1,400 (>50%) of the world’s remaining Grevy’s zebras. The county also has a stable lion population estimated at 5.3 adult and subault lions per 100 km2 , 17. His2  such thae 1960’s  Fig 1)..2 [2 in e 1960’s , 20, 21.Equus quagga) abundance increased 5-fold, providing an abundant food supply for recovering predator populations. Droughts caused widespread livestock and wildlife mortality that provided abundant carrion, and periods of high rainfall resulted in taller grasses and better cover for hunting predators. Although a century of fire suppression has caused large scale landscape transition from grassland to bush [Alcephalus buselaphus) and eland (Tragelaphus oryx), were due primarily to increasing predation pressure. A recent study by Ng’weno et al. [2 fenced conservancy in Laikipia with a very high lion density (~24 lions/100 km2), and that increasing bush encroachment contributed to the decline.Since the late 1990’s, many large ungulate populations, including Grevy’s zebra, have been declining in Laikipia County , whereas to bush , Georgia to bush  surmised2 fenced rhinoceros reserve neighboring Laikipia County. In 2000, LWC introduced lions to a naïve community of large ungulates; within three years, population counts showed a 17% and 19% reduction in Plain’s and Grevy’s zebras, respectively . In an analysis of LWC lion scat, Rubenstein [Rubenstein  explicitbenstein  found thThese observations have raised concerns about the impact of lion predation on the endangered Grevy’s zebra and the potential for contradictory conservation goals: successful lion conservation may impede successful Grevy’s zebra conservation in Laikipia County and elsewhere. The studies above, however, either were unable to establish a causal link  or were To answer this question, we evaluated lion predation on Grevy’s and Plain’s zebras across a large, mixed-use landscape in Laikipia County using satellite telemetry to track lions and zebra movements, and to locate and identify lion kills. We used random gas models to address the question of whether lions are preying on Grevy’s zebra more than expected due to chance alone and to assess the likelihood that lions are negatively influencing Grevy’s zebra numbers.2: We conducted our study in Laikipia County, Kenya  are concentrated in the north. Our study focused on a 2,770 kmLaikipia , a largeLaikipia ; savannaia tenax .This study was reviewed and approved by the Office of the President of the Republic of Kenya and the Kenya Wildlife Service , and relevant research committees at University of California, Berkeley, University of California, Santa Cruz and Princeton University. The lion collaring protocol was reviewed and approved by the Animal Care and Use Committee, University of California, Berkeley, Protocol No. R191 (to LGF) and the Institutional Animal Care and Use Committee, University of California, Santa Cruz Permit No. WILMC1402 (to CW and TW). Lion anesthesia: lions darted with a combination of Medetomidine and Ketamine. The zebra collaring protocol was reviewed and approved by the Institutional Animal Care and Use Committee, Princeton University, Protocol No. 1845 (to DR). Zebra anesthesia: Kenya wildlife Service Veterinarians darted using M99. All species in this study are considered protected species under Kenyan Law. Population estimation of Grevy’s and Plains zebras was conducted using non-invasive line transect surveys and were conducted on private ranches.rocuta crocuta) regularly appropriate kills from each other [Lions are almost entirely nocturnal in Laikipia County and lion kills are rarely found due to dense bush. Formerly, tracking circling vultures was a reliable means of locating carcasses but the collapse of Kenya’s vulture populations due to predator poisoning , 16 reduch other , 31, as Zebras were darted from a vehicle using tranquilizer darts containing M99 (Princeton University IACUC Protocol No. 1835) under the supervision of a Kenya Wildlife Service veterinarian. Zebras were collared using custom-made GPS-GSM collars  and movement and ranging data were recorded at 15-minute intervals. Grevy’s zebra movements were monitored in 2007–2008 and Plain’s zebras were monitored in 2010–2011.We used line transect surveys to estimate density and abundance of Plain’s and Grevy’s zebras. One hundred and seven 2-km transects and seventy-one 3-km transects were laid out systematically along existing roads in the southern and northern parts of the study area, respectively. Approximately 70 transects were located in bushland and the remaining surveys were in savannas and open woodland. It is usually inadvisable to use roads for line transect surveys , 33 if tSouthern transects were surveyed once in June 2014 and again in January 2015. Northern transects were surveyed twice during January 2015. All surveys were conducted in the morning between 0630 and 1000 and late afternoon between 1630 and 1830. Two observers, positioned on the roof of a vehicle at approximately 3 m height, recorded all individuals and groups of zebras encountered as part of a larger, multi-species survey. We measured observer-to-animal distances using laser rangefinders , and transect bearing and angle between animal and observer with digital compasses . Perpendicular distance to the transect was calculated using the angle between transect and animal and radial distance from observer to animal.We used DISTANCE 6.0 software  to analy1 moving at a velocity of v1 and predators with density p2 and velocity v2, the expected number of encounters that result in a predation attempt by a single predator unit, Epred, is a function of prey and predator densities, velocities, the distance D between predator and prey that defines an attempted predation encounter, and time t as follows:2, velocities are expressed as km/12 h, distance for an encounter is expressed as fraction of a km, and time is in days. The expected kill rate, Ekill given an encounter is simply Epred times the proportion of attempted predation encounters that result in kills (S):A prey species is considered preferred when it is taken more than expected based on a random distribution of encounters conditioned on the abundance of predator and prey species , 36. We pred and Ekill, we used a bootstrap approach. Density of lions was estimated as the number of collared lions (n = 21) in a 2,105 km2 area defined by the union of all 95% minimum convex polygon home ranges for lions, or 0.01 lions/km2. We assumed that each collared lion was an independent hunting unit. Grevy’s and Plain’s zebra densities were estimated from line transect surveys as the cluster density since lions were expected to encounter a group of zebras and kill a single member of the group when an encounter resulted in successful predation. In the simulations, lion density was treated as a constant. Zebra densities were randomly chosen from normal distributions with observed density and standard deviations, truncated at 0.To estimate Ekill, we used values of 10%, 15%, 20% and 25%. Each simulation was run 10,000 times and a mean, SD and 95% CI was calculated for each combination of parameters. We then compared the simulation kill rates over the course of the study to the estimated kill rates based on field data for the number of potential feeding sites, the proportion of actual feeding sites among potential feeding sites and the proportion of Plain’s and Grevy’s zebras among recovered kills.To estimate velocities, we used hourly movements from satellite telemetry fixes  collecteBetween 27 November 2013 and 1 January 2015, we collared 21 female lions in 10 prides  and trac2 (95% CI = 1.61–2.42) and individual density at 15.94/km2 (95% CI = 12.82–19.82). We estimated the density of Grevy’s zebra groups at 0.30/km2 (95% CI = 0.17–0.53) and individual density of 0.71/km2 (95% CI = 0.38–1.34). Coefficient of variation was higher for Grevy’s zebra compared to Plain’s zebra due to small sample sizes.We conducted 387 line transect surveys totaling 904 km of survey effort, and observed 697 Plain’s zebra groups and 68 Grevy’s zebra groups . Mean grBos Taurus: 12.6% of kills). Only 2.0% of estimated kills were Grevy’s zebra. This represents an estimated 17.6 Plain’s zebra and 0.7 Grevy’s zebra killed per lion during an average 226.5 days that the 21 lions were followed, or 28.4 Plain’s zebra and 1.2 Grevy’s zebra killed per lion per year. The study area contains a full complement of predators, and lions are known to take prey from hyenas, leopards and cheetahs. Because our methods could not distinguish prey killed by lions from prey scavenged by lions, predation rates could potentially be lower.We determined 3,993 instances during which a lion moved less than 50 m over 3 or more hourly fixes  during 4,756 days of telemetry data. We investigated >3,000 potential lion feeding sites and located 768 kills , the modeled estimate of predation was significantly lower than field-based estimate of predation . For ninExpected predation rates by lions on Grevy’s zebra generated using the random gas model were significantly higher than the field-based estimates for all but the most conservative scenario  of cases. In comparison, predation on Plain’s zebras is as expected (9 scenarios: 56%) or less than what would be expected due to chance alone (6 scenarios: 38%). Observed predation on Grevy’s zebra was consistently less than expected for all model scenarios, equaling the lower limit of the confidence interval only for the most conservative scenario . As modelled encounter distance and success rate increased, the disparity between observed and expected predation rates also increased, with the random gas models predicting significantly more predation than observed in the field for both subspecies. Because predicted selective predation by lions on Grevy’s should show a greater rate of encounters and therefore a larger number of observed kills compared to random encounters, our data indicate that lions are not preying selectively on Grevy’s zebras in Laikipia County.Our results support the hypothesis of Georgiadis et al.  that lioThe random gas model has been applied widely as a null hypothesis to model encounter rates within and among species , 43, 44.Rubenstein  modeled Threats to Grevy’s zebras  include:S1 TableDates that collars were operational, number of sample days, hourly distance traveled (SD), and daily distance traveled (SD) for female lions, Plains zebra and Grevy's zebra. Lions with numbers were recaptured more than once and collar either changed or repaired during study.(DOCX)Click here for additional data file.S1 AppendixHourly movement data for African lions, Grevy’s zebras and Plains zebras used in simulations of encounter rates.(XLSX)Click here for additional data file.S2 AppendixThe compatibility of temporally disconnected movement data.(DOCX)Click here for additional data file.


Currently, only 58% of the São Paulo tree flora currently have at least one barcoding sequence available. However, these species represent on average 89% of the trees in São Paulo state forests. Therefore, conservation-oriented and ecological studies can already benefit from DNA barcoding to obtain more accurate species identifications. We present which taxa remain underrepresented for the São Paulo tree flora and discuss the implications of this result for other species-rich tropical regions.DNA barcoding helps to identify species, especially when identification is based on parts of organisms or life stages such as seeds, pollen, wood, roots or juveniles. However, the implementation of this approach strongly depends on the existence of complete reference libraries of DNA sequences. If such a library is incomplete, DNA-based identification will be inefficient. Here, we assess if DNA barcoding can already be implemented in species-rich tropical regions. We focus on the tree flora of São Paulo state, Brazil, which contains more than 2000 tree species. Using new DNA sequence data and carefully assembled GenBank accessions, we assembled 12,113 sequences from ten different regions. The ITS,  Altitude varies from sea level to nearly 2800 m asl. Climate is mainly characterized by a marked rainy summer and dry winter (oC (http://www.ciiagro.sp.gov.br). São Paulo is one of the Brazilian states with the highest degree of knowledge in terms of its flora. It shelters 7528 seed plants, mainly within the Atlantic Forest and Cerrado domains, two important biodiversity hotspots of plant and animal endemism (São Paulo State in south-eastern Brazil (19º47’-25º19’S latitude and 44º10’-53º06’W longitude) has a total area of 248 thousand kmy winter . Howeverendemism .Ficus, Coussapoa, Spirotheca and Oreopanax. Hereafter, we will refer to this checklist as the SP tree flora. This list is based largely on the Brazilian Flora Checklist project  in São Paulo, for which we found vouchers only for the state of Rio Grande do Sul in the speciesLink network (http://splink.cria.org.br).We compiled a list of all native and naturalized woody species occurring in forests of the state of São Paulo, including trees, tall shrubs and arboreal species of palms, ferns, cactus and bamboos. We did not include woody lianas, but we did include woody hemi-epiphytes species of  project  which pr project , which cTable S1). These surveys were compiled by Cerradão), obtained using the same search methods. Most of surveys (53%) had total sampling effort ranging from 1 to 10.24 ha and a cut-off criterion of diameter at breast height ≥ 4.8-5.0 cm. Overall, the 135 surveys contained abundance records from around 174 ha of natural forests and 269,000 individuals. These surveys were used to calculate the proportion of individuals in each community belonging to species with DNA barcodes currently available. The distributions of these proportions across sites (and their descriptive statistics) were taken here as a measure of the extent to which DNA barcoding can be used to assist the identification of tree individuals occurring in São Paulo forests. This assessment was carried at species, genus and family levels. Records determined as affinis were considered only at genus level, but species determined as confers were attributed to the suggested species.The abundance of tree species in São Paulo forests was obtained from 135 published tree community surveys , the semi-deciduous seasonal forest, white-sand forest , and the rain forest over clayey soils. In addition to the tagged trees inside each permanent plot, we also sampled the most abundant shrubs that usually did not reach the 4.8 cm diameter at breast height . In a second moment, additional sampling campaigns were carried out aiming to collect samples of species from orders and families that were not present in the four 40.96-ha plots. We performed rapid surveys in 40 forest fragments in 38 counties of São Paulo, collecting fertile specimens in the forest border and interior. Species identification was validated by consulting several specialists and taxonomic revisions. Vouchers for all species sampled were deposited at ESA and RB herbaria  forest plots of the ‘São Paulo Permanent Forest Plots’ project . TogetherbcL and matK for all families and the ITS region specifically for Myrtaceae and Melastomataceae, two species-rich families in the SP tree flora for which we had low amplification success for both plastid barcodes. Our choice of markers was related to an ongoing initiative funded by the state government to produce a complete reference library of plant DNA barcodes for the entire state of São Paulo, starting with the two core plastid barcodes. The DNA sequences were obtained using classic procedures of DNA extraction  and the ‘Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis’ (IBAMA - License 14BR013854).We assembled DNA sequences for traction , for allrbcL, matK, the ITS region (i.e. 18S|ITS1|5.8S|ITS2|26S) and psbA-trnH  for the valid binomials and synonyms of the SP tree flora recognized by the Brazilian Flora Checklist project. Our search included only accessions deposited until 31 December 2015. It was conducted using the ‘organism’ search field and was restricted to vascular plants. Only accessions verified by GenBank were considered. For each verified accession retrieved, we extracted the information on the date of submission, molecule type, the origin of the specimen, and the gene to which the accession referred to. We then classified genes as barcoding and non-barcoding plant DNA regions. The majority of studies on plant DNA barcodes concentrate on a/(1 + cexp(btime)); see a, so that we could estimate the date when the accumulation curves saturate according to the best logistic fit to data. We fitted functions using non-linear least squares regression  the proportion of barcodes not available, (ii) the proportion of threatened species without barcodes, and (iii) the proportion of endemic species without barcodes (see details in Table S3). We summed these three proportions to calculate a priority index varying between 0 and 3. The higher the index, the greater the priority for future sequencing efforts. To visualize this result, the priority index was mapped upon the phylogeny for the SP tree flora. This phylogeny was obtained at family-level using the stored megatree R20120829 available from the phylomatic website (http://phylodiversity.net/phylomatic)Within each family and genus, we calculated the difference between the overall proportion of species in the SP tree flora and the proportion of species with DNA barcodes. We fitted a Cauchy distribution to the values of difference between these proportions using maximum likelihood and calculated the 95% and 90% confidence intervals to determine which groups had an under or overrepresentation in the DNA barcode library of the SP tree flora. The Cauchy distribution, which is a special case of the Student’s J):To assess the representativeness of each barcode in respect to the São Paulo tree flora at different taxonomic levels, we implemented the following procedure. We calculated the abundance of sequences at order, family, genus and species taxonomic levels and then computed the Pielou index . The response variable was the count of barcoding sequences available per species, which varies from 0  to 10 , and presented a skewed distribution with much more small than large counts per species. This variable was regressed on species geographical distribution, namely non-endemic, Eastern South-American endemics, regional endemics and local endemics. The significance of this regression model was tested using a Likelihood ratio test. All analyses conducted were performed using R .Table S4). It represents around 85%, 68% and 48% of the families, genera and species of native trees recorded for the Atlantic Forest and Cerrado domains together  from 36 orders, 107 families and 508 genera ). By the end of 2015 GenBank contained nearly 30,000 accessions for the SP tree flora, of which 39% corresponded to sequences of the ten DNA markers considered here (Table S5). Few studies made great contributions to this increase  for 609 species , among which more than 80 are new species entries in the NCBI taxonomy database. This is by far the largest sequencing effort ever carried out for SP tree species. Today, there are currently ca. 12 thousand DNA barcodes available for the SP tree flora  were so scarce  that these markers will not be discussed hereafter. We found that matK and rbcL had the highest evenness across taxonomic levels. In terms of evenness, trnL also outperformed the ITS region. None of the barcodes had sequences for more than 40% of SP tree species (rbcL+matK (33%), rbcL+ITS (21%), rbcL+psbA-trnH (15%) and matK+psbA-trnH (15%), but they are slightly higher for three-barcodes combinations, such as rbcL+matK+ITS (18%) or rbcL+matK+psbA-trnH . The only family lacking barcode information is Schoepfiaceae, represented by Schoepfia brasiliensis, for which we did not manage to amplify neither rbcL nor matK. Regarding the threat and endemism status of species without barcodes, other groups come out as priorities for future sequencing efforts: Dichapetalaceae, Escalloniaceae, Hypericaceae and Pentaphylacaceae (Table S3). They are all rare in SP, have high proportions of threatened and endemic species, but lack DNA barcode sequences.Genera that have proportionally fewer species with DNA barcodes than average were Lychnophora ericoide, Handroanthus impetiginosus and Tabebuia aurea).Although the number of accessions has increased in an exponential fashion, the accumulated number of taxa with barcodes has increased much more slowly. Currently, over half (58%) of SP tree species have DNA barcode information. If current accumulation rates remain the same, 100% of its families, genera and species will have at least one barcoding sequence available by 2026, 2033 and 2040, respectively . Few speTaking into account the abundance of the species with at least one DNA barcode currently available, they correspond on average to 88.9% of the tree individuals  across 135 surveys of São Paulo state forests. Because these sites had on average 1.5%  of their individual unidentified at species level, this proportion may be underestimated. When considered at genus and family levels, this proportion reaches 99.0%  and 99.5% , respectively.p < 0.0001). Widespread species had on average more barcodes available than Eastern South-American, regional and local endemic species (in decreasing order of number of barcoding sequences per species). Regional and local endemics also presented significantly less rbcL and matK sequences (12.9%) than other species . The same outcome was found when analysis was conducted considering only the top five DNA barcodes .The SP tree species list contains 237 local endemics (11%) and 493 regional endemics (24%). The availability of DNA barcoding data was strongly related to species geographical distribution  suggest that 71-88% of the individuals from Atlantic and Cerrado forests in other regions of Brazil, Paraguay and Argentina already have sequences in the current DNA barcoding library. The exception is Northeast Brazil where this percentage is only 41%.In species-rich forests, the identification of all individuals in a given community is often a difficult and subjective task (P > 1 in Table S3) would therefore provide many sequences for taxa with high conservation relevance. As shown here, trees endemic to SP were indeed the ones with fewer barcoding sequences.Defining which taxa should be prioritized in future sequencing efforts is not easy. Here, we defined a family-level priority scale based on the proportion of species without barcodes per family and on how many of them were threatened or endemic. This definition was first thought in the context of using DNA barcoding in assessments of conservation status in biodiversity surveys . HoweverrbcL and matK), and many taxa will need the combination of two or more barcodes to guarantee accurate identifications . Complementarily, the ITS and psbA-trnH regions could be amplified in selected clades that present low discrimination rates using only rbcL and matK (trnL intron would be another important marker to increase focus on because a short region on this marker (the P6 loop) is often recommended for plant diversity surveys based on degraded DNA from environmental samples (Having barcodes for all species does not necessarily guarantee a precise identification at species level. Within some genera, species cannot be resolved using only core DNA barcodes (i.e. New sequencing technologies are becoming rapidly available and these have greatly facilitated the sequencing of complete plastid genomes in plants (In the past five years, it seems that DNA barcoding has fallen out of interest, probably due to the exposition of its limitations , or to t


A novel breathable piezoelectric membrane has been developed by growing zinc oxide (ZnO) nanorods on the surface of electrospun poly(vinylidene fluoride) (PVDF) nanofibers using a low-temperature hydrothermal method. Significant improvement in the piezoelectric response of the PVDF membrane was achieved without compromising breathability and flexibility. PVDF is one of the most frequently used piezoelectric polymers due to its high durability and reasonable piezoelectric coefficient values. However, further enhancement of its piezoelectric response is highly desirable for sensor and energy-harvester applications. Previous studies have demonstrated that piezoelectric ceramic and polymer composites can have remarkable piezoelectric properties and flexibility. However, devices made of such composites lack breathability and some present health risks in wearable applications for containing heavy metals. Unlike other piezoelectric ceramics, ZnO is non-toxic material and has been widely used in many applications including cosmetics. The fabrication of ZnO@PVDF porous electrospun membrane involves a simple low-temperature ZnO growth in aqueous solution, which does not weaken the polarization of PVDF created during electrospinning in the high electric field. Interest in wearable piezoelectric materials has grown tremendously due to the increasing need for powering mobile devices, achieving sustainable operations, and measuring long-term biometric data ,2,3. PieChemical resistivity of PVDF also brings forth stability and safety for use next to human skin. To select piezoelectric ceramics to be embedded in PVDF matrix, the potential health hazard is one of the most important factors to be considered ,21. LeadPiezoelectric polymers can be made into thin films or fibers. Fibrous materials are both breathable and flexible, and therefore more suitable for wearable applications than thin films. Also, one-dimensional PVDF fibers can have higher piezoelectric energy conversion efficiency than films ,39. HoweHere we present a piezoelectric nanofibrous material with improved piezoelectric responses by growing ZnO nanorods onto the PVDF fiber surface (ZnO@PVDF) through a facile hydrothermal process. We have also fabricated a truly breathable and flexible fibrous nanogenerator with conductive textile electrodes without impermeable casing.Mw = 180,000, Millipore Sigma, Burlington, MA, USA) was prepared with N,N-dimethylformamide  and acetone . The PVDF solution was prepared by adding PVDF pellets to the DMF-acetone solvent mixture (7/3 v/v), and then stirred with a magnetic stir bar on a hotplate at 70 °C, 150 rpm for three hours. The PVDF solution was withdrawn into a syringe with spinneret needle gauge size 23 and was then pushed by a syringe pump  with a rate of 1.0 mL/h. A high voltage of 14 kV was applied by a DC power supply  to the needle. Fibers were accumulated on a grounded 100-mm-diameter collector rotating at 200 rpm. The distance between the cylindrical collector and spinneret was 10 cm. A Teflon tube was used to connect the syringe and the needle, and the needle was moved laterally with a stroke of 18 cm and approximately four strokes per minute to obtain wide and uniform nanofiber mats.PVDF nanofiber mats were fabricated by electrospinning. The polymer solution of 20 wt % of PVDF  was dissolved in 150.0 mL of isopropyl alcohol, and the solution was stirred vigorously at 85 °C for 15 min. Then, trimethylamine  was added dropwise to the solution which subsequently became clear. The solution was then stirred again at 85 °C for 10 min and incubated at room temperature without stirring for three hours. Isopropyl alcohol (600 mL) was added to dilute the solution to 10 mM before use.Hexamethylenetetramine  was dissolved in 400 mL of deionized (DI) water, and the solution was stirred for 10 min. Zinc nitrate hexahydrate  was added to this solution, which was followed by stirring for 24 h. All processes were performed at room temperature. DI water was added to 10-fold dilution before use .PVDF nanofiber membrane was placed in between two Teflon frames  for structural stability. The seeding procedure was done in three rounds comprising of immersing in seed solution and curing in 60 °C oven. For the first round of seeding, the membrane was immersed into the seed solution (10 mM) for 20 min, then cured in 60 °C oven for 20 min. For second and third rounds, immersion was for two minutes, and curing was the same 20 min. After the seeding process, the PVDF nanofibers in the Teflon frame was dipped into 400 mL of growth solution , and placed in 60 °C oven for 6 h. The sample stayed in the inert oven for gradual cooling down to the room temperature, which typically lasted for 11–13 h. The samples were then rinsed with running DI water for five minutes and dried in air.The piezoelectric testing module was assembled by sandwiching the piezoelectric nanofiber mat between two electrodes made by either conductive fabric  or aluminum foil.Piezoelectric performance of the electrospun samples was evaluated by periodic tensile testing in a customized setup with a motorized actuator controlled by a controller and a module , a pressure sensor , electrometer , multimeter  and programmable DC power supply . The testing head movement was set to 1 Hz, and impact pressure was set to 0.10 MPa which is within the human foot pressure range of 0–0.20 MPa ,46,47, aField-emission scanning electron microscope (FESEM) images were taken using LEO 1550  with an accelerating voltage of 3 keV. To extract the detailed geometry with better focus and to reduce the charging effect under FESEM, the samples were partially sputter coated with palladium/gold. The average PVDF fiber diameter and ZnO nanorod length and diameter were determined by FESEM images over 30 fibers with the ImageJ software, NIH, Bethesda, Rockville, MD, USA .Kα radiation (λ = 1.54 Å) at a 0.02° scanning step and an operating voltage of 40 kV and current of 40 mA are used.Crystallography of PVDF nanofibers and ZnO nanorods was examined by X-ray diffraction (XRD) with the powder diffractometer , where Cu-−1  was performed at room temperature to evaluate the polymer crystalline phase. FTIR spectra were collected with 16 scans and a resolution of 4 cm−1.Fourier-transform infrared (FTIR) spectroscopy from 4000 to 650 cmThermal gravimetric analyses (TGA) were used to determine the amount of ZnO added to PVDF nanofibers  by heat removal of PVDF. Samples were heated up from room temperature to 990 °C with the rate of 10 °C/min on a ceramic pan under a nitrogen ambient.The water vapor permeability (WVP) was measured with an upright cup method according to BS 7209 ,50,51. TPVDF has a high piezoelectric coefficient among piezoelectric polymers and is a semi-crystalline polymer with up to five different crystal phases  . Two mosThe nanostructure of PVDF nanofibers and ZnO nanorods were first examined under FESEM. As described in the experimental section, the ZnO nanorods were grown on the PVDF fiber surface by the hydrothermal growth method. The FESEM image in −1 , 766 cm−1 , 795 cm−1 , 855 cm−1 , and 976 cm−1  [−1  and 1279 cm−1  got stronger, and peaks corresponding to α crystalline got weaker. Hydrothermal growth of ZnO nanorods did not depolarize PVDF fibers, and similar FTIR spectra of nanofibers confirmed this before and after the growth process. The maintained intensity of β crystalline peaks indicates that PVDF polarization does not relax during hydrothermal growth. Furthermore, the β-phase percentage can be quantified using the following equation:−1, and 2·mol−1, respectively [FTIR spectra were studied to analyze the PVDF crystalline phases under the electrospinning and hydrothermal growth process. FTIR spectra of the PVDF polymer pellets, electrospun PVDF nanofibers, and ZnO@PVDF nanofibers are compared in rmation) ,60. Afteectively ,60,61. Tectively . From thThe amount of ZnO added onto PVDF fibers was evaluated by TGA . It was 2, a frequency of 1 Hz and pressure of 0.10 MPa, emulating foot pressure during walking.Aluminum foil and conductive knitted fabric were used as electrodes for comparison. Four types of nanogenerators were made with two types of piezoelectric nanofibrous membranes, PVDF and ZnO@PVDF, and two types of electrodes, that is to say, aluminum foil and conductive knitted fabric. Open-circuit voltages were measured to evaluate the two electrode materials. Open-circuit voltages for aluminum foil electrodes were found to be higher than those for conductive knitted fabric electrodes for both PVDF and ZnO@PVDF. Also, the variation of measurements was larger for aluminum foil than for the conductive knitted fabric. A possible explanation for the conductive fabric electrodes giving more stable and reproducible results is that it provided better adherence between the piezoelectric membrane and the electrodes. We opted not to use any additional casing to press adherence of electrodes and piezoelectric membrane layers to maintain the best flexibility and breathability, and thus the air gap between the layers can exist due to the lack of external forces. The flexibility and stretch-ability of the materials determine the amount of air gap created. Such properties can be measured with the Fabric Assurance by Simple Testing (FAST) system . The benSince conductive knit electrodes provided more consistent piezoelectric responses than aluminum foil, conductive knits were used to construct breathable nanogenerator made of as-spun PVDF and ZnO@PVDF for further evaluation.I-V sharpness fitting. Closed-circuit currents of the nanogenerators with different resistive loads were compared, and the closed-circuit voltages and the power density were derived accordingly. As can been seen from −2·day−1), M the loss in mass (g), t the time duration (h), and A the area of the exposed test fabric which is equal to the internal area of the dish (m2). The results were averaged from three specimens over 60 h.The breathability of piezoelectric fibrous nanogenerators, as well as that of the constituent materials, were evaluated by measuring the water vapor permeability for the nanogenerators, which consisted of an electrospun PVDF or ZnO@PVDF membrane sandwiched by two layers of conductive knitted fabrics. An upright cup method is employed in accordance with the BS 7209 standard. A cotton woven fabric was also tested at the same time for a benchmark. The water vapor permeability (WVP) was calculated using the following formula:From High piezoelectric response, breathability, and flexibility are essential to nanogenerators for wearable applications. The present study demonstrated that breathability and flexibility of the fibrous nanogenerators can be achieved by assembling a layer of an electrospun piezoelectric nanofibrous membrane with two layers of conductive knitted fabric and without the use of rigid plastic casing. It further showed that the piezoelectric responses of PVDF fibrous nanogenerators can be significantly enhanced (up to 2.3 times regarding open-circuit voltage) by growing ZnO nanorod onto the PVDF fibers via a low-temperature hydrothermal process which does not weaken the polarization of PVDF created during electrospinning.Our nanogenerator performance was compared with the previous methods in


Evodiamine, an alkaloid contained in traditional Asian herbal medicines that have been used for hundreds years, is interesting due to its cytotoxic effects against many cancers. We examined the effect of evodiamine on the cancer stem cell (CSC) population and the bulk cultured cancer cells (BCC) of colon cancers to examine the double targeting effect. We found that three colon cancer cell lines’ BCC and CSC are effectively targeted by evodiamine. Evodiamine was able to suppress BCC proliferation and induce apoptosis of the cells captured in G2/M phase, as previously reported. However, evodiamine did not cause the accumulation of CSCs at a certain stage of the cell cycle, resulting in the elimination of stemness through an unknown mechanism. By analyzing the expression of 84 genes related to CSCs in two colon cancer cell lines’ CSC, as well as performing further informatics analyses, and quantitative RT-PCR analyses of 24 CSC genes, we found that evodiamine suppressed the expression of the genes that control key signaling pathways of CSC, namely, WNT and NOTCH signaling, to lead CSC elimination. These results suggest that evodiamine should be further developed for targeting both BCCs and CSCs in colon cancers. Over the last half century, intense efforts led by developed countries have been devoted to cancer research, many new anticancer therapeutic strategies were developed, and there have been significant improvements in the prognosis of several subtypes of cancer. However, the mortality of most cancer types is still very high, leaving the disease one of top causes of death world wide . At leasA small population in the tumors, “cancer stem cells or CSCs”, is also believed to have pivotal roles in cancer initiation, resistance and metastasis . IronicaTo find druggable pathways that target CSCs and BCCs together, we performed siRNA screening and revealed several potential targets . SeveralColorectal cancer is the 3rd most common cancer type worldwide, and the incidence is believed to be related to diet. The mortality is very high due to late diagnosis resulting in metastasis and resistance to conventional therapies.Evodiae fructus, which has been used for long time in Asian medicine for symptoms including digestive problems [Evodiamine is a main component of plant problems . The antproblems ,12. Howeproblems , and we We tested the potential for evodiamine as a colon CSC-targeting agent and compared the effect in BCCs to determine if the compound can be used to eliminate both populations of colon cancer cells. Evodiamine was an effect targeting agents for both BCC and CSC inducing apoptotic cell deaths and reduced the stem cell potential in vitro and in vivo. Biochemical and genetic mechanistic study revealed WNT and Notch pathway, which are essential for cancer stem cell signaling, are primarily modulated by evodiamine in CSC of two cell lines suggesting evodiamine as a strong candidate to target colorectal BCCs and CSCs together.To examine if evodiamine could be used to treat the colon BCCs and CSCs together, we tested if evodiamine suppressed the growth of both the BCCs and CSCs of colon cancers using three colon cancer cell lines . In BCC culture, the cells were maintained in the medium supplemented with FBS or without serum and attached on the culture dish. The cell growth of all three cell lines in both the BCC and CSC conditions was effectively inhibited by treatment with evodiamine at concentrations between 0.1 and 1 μM. At the low concentration, the serum-free condition cultured cells were more resistant than the FBS-supplemented cultured cells, but this resistance was reversed at higher concentrations in the SW480 and HCT15 cells. The growth of CSC spheres was also effectively inhibited at concentrations lower than 1 μM in all three tested cell lines A. The miTo examine whether the inhibitory effect on BCCs and CSCs occurs through G2/M arrest, as previously reported, we performed cell cycle analyses on two cell lines, HT29 and SW480. In both conditions, the dying population increased at 48 h of treatment in a dose-dependent manner A, and thSelf-renewal activity is a surrogate marker for colon cancer CSC potential, and the population with this activity can be quantified using limiting dilution analysis (LDA) . We testThe different patterns of cell cycle changes in the CSCs and BCCs suggest the molecular mechanism through which evodiamine induces cell death in the two cell types may be independent of each other. The BCCs were captured at G2/M checkpoint and may undergo apoptosis as we published in breast cancer . However2 profile PCR array, Qiagen). The changes in the expression in 84 genes related to CSC biology were screening together  have been experimentally demonstrated to interact with NOTCH1 and five genes  that interact with WNT1 were identified. In addition, six genes  for NOTCH1 and one gene (NANOG) for WNT1 were expected to functionally interact. Notch and WNT signaling are the pivotal players of tissue stem cells and cancer stem cells [To determine the common mechanism targeting these two cell lines’ CSCs, we further analyzed the data and the selected genes for which expression was commonly increased and decreased after this mild treatment of evodiamine in both the HT29 and SW480 cells lines A. Since em cells ,20,21,22em cells ,25,26,27- PCR array result, we hypothesized that evodiamine suppresses NOTCH and WNT signaling. To test this hypothesis, we further analyzed time dependent changes of gene expression, related to main CSC signaling pathways. A mild concentration of evodiamine (200 nM) was treated to HT29-derived CSCs for 48 h and 72 h, and the expression of NOTCH signaling  and WNT signaling (LRP5 and LEF1) genes was assessed. In addition, other genes related to FGF (FGFR3) signaling and Hh  with CSC markers (CD133 and SCD1) were also tested  biological efficacy is already warranted by empirical evidence for hundreds years. Second, a range of dose for safety and toxicity can be estimated by accumulated knowledge’s. Herbs containing evodiamine have been used for a long time in East Asia for the treatment of a range of conditions, including digestive problems and pain . RecentlAs reported in BCCs in many tissues and CSC in breast cancer, the CSCs of colon cancers died due to apoptosis at concentrations of evodiamine between 0.2 and 1 μM. Compared to the certain size (over 100 μm) of sphere numbers, which decreased very little in response to 200 nM evodiamine A, the CSWe finally tried to analyze the time-dependent changes of CSC molecules using RT-PCR in HT29 cells that were treated for 48 h and 72 h with 200 nM drug. At this concentration, we started to see the CD133 and SCD1 expression change, and this change was accompanied by a decrease in Notch1, Jagged, and LRP5. The expression of these genes was further decreased as CD133 and SCD1 decreased. However, all other genes were rather activated at 48 h. It seems this is due to the suppressed Notch and WNT signaling in the cells, may lead feedback effects to the suppressed CSC signaling. The following decrease of most other genes at 72 h may be related to the CSC specific elimination already started (considering CD133 and SCD1 at this points).Collectively, we demonstrated that evodiamine had an interesting potential for simultaneous targeting colon CSCs and BCCs. The molecular mechanism of BCC targeting may be similar to the previously known mechanisms for other tissue cancers, which arrests the cells at the G2/M checkpoint and induces apoptosis. However, it primarily suppresses expression of WNT and NOTCH signaling genes, essential for CSC stemness or/and the maintenance. We therefore suggest that evodiamin’s interesting dual targeting effect might act through CSC-specific signaling in colon CSCs, which needs to be investigated further for development for clinical use.2. All plastics culture wares were purchased from SPL (Korea). For cell counting, the cells in 96 wells were stained with Hoechst 33342 (Sigma) and imaged using a Cytation™ 3 . The number of CSC spheres with a diameter over 100 μm was counted after taking pictures with Nikon microscope. Evodiamine was purchased from Sigma (USA). The antibodies were acquired from Cell Signaling Technology .The human colon adenocarcinoma cell lines, HT29, HCT15, and SW480 were purchased from American Type Culture Collection . The cells were cultured in Dulbecco’s modified Eagle’s medium or RPMI1640  supplemented with 10% fetal bovine serum  and 100 units/ml Anti/anti (Gibco). CSCs were enriched in DMEM/F-12  supplemented with EGF  basic fibroblast growth factor , and B27 supplement  on in house prepared ultra low adhesive culture plates, as described previously . All cel2 First Strand Kit  and applied to RT2 Profiler™ PCR Array Human Cancer Stem Cells  to examine 84 CSC related target genes together in Stepone Plus . The data were further analyzed with Strings database (https://string-db.org/cgi/network.pl) [Total RNA was collected from CSCs and converted to cDNA using RTwork.pl) .Total RNA was also extracted from collected CSCs and BCCs with TRIsure  and converted to cDNA with SuperScript II (Thermo Fisher Scientific). qRT-PCR was carried out ,4 using Limited dilution assay to determine self-renewal active cell density was performed based on previously published methods . Briefly6 alive cells were subcutaneously implanted in dorsal flank of in NOD/SCID . The size of the tumor was measured with calipers, calculated by width × width × length × 1/2 every other days. The weight was also measured every other day. In each group, five mice were used. All mice were maintained in specific pathogen free facilities on a 12 hours light/12 hours dark cycle. At the endpoints, the mice are euthanized with excess CO2 gas.All experiments were pre-approved by IACUC of Sookmyung Women’s University. Alive BCC cells after treated with Evodiamine (1 μM) for 24 h were counted with trypan blue exclusion assay and 1 × 10Dissociated single cells were treated with 250 μL 0.6% Triton X-100 containing 50 μg of RNaseA for 1 h at room temperature and stained by 5 μL of 1 mg/ml Propidium iodide. An assay was performed by Calibar  and the data was analyzed by Flowjo software .Spheres were fixed using 4% formaldehyde for 20 min and washed with PBS. For dehydration, the samples were immersed in ethanol and displaced into hexamethyldisilazane (Sigma-Aldrich). For mounting the samples, the samples were sputter coated with gold on aluminum stubs with adhesive carbon tapes. The samples were observed by scanning electron microscope, JSM-7600F .3VO4, 1 mM NaF, and 20 mM β-glycerophosphate) and sonicated. The cleared lysates were resolved in SDS-polyacrylamide gel by electrophoresis and then transferred to a PVDF membrane . The membranes were blocked by 5% non-fat dry milk in a buffer containing 20 mM Tris pH 7.4, 150 mM NaCl and 0.1 % Tween-20, then probed with primary antibodies and secondary antibodies with horse radish peroxidase. The signals were detected Supersignal West Femto , or Novex . All original raw western blotting images are seen in The BCCs and CSCs were treated with evodiamine for specified time and the cells were harvested in the lysis buffer containing Tris-Cl pH 7.4, 1% NP-40, 1% triton X-100, 0.25% sodium deoxycholate, 150 mM sodium chloride, 10% glycerol, protease inhibitors , and phosphatase inhibitors (1 mM NaEvodiamine suppresses WNT and NOTCH signaling in CSC and can be used for dual targeting agents for BCC and CSC of colon cancer.


Background: Maternal colonization of Group B streptococci (GBS) during pregnancy is an important risk factor for neonatal morbidity and mortality. The aim of this study was to determine the prevalence and serotype distribution of GBS isolated from a cohort of pregnant women in the Western Cape, South Africa.Methods: Two ano-vaginal swabs were collected from 301 women at 28–37 weeks of gestation. Participants were recruited from four different antenatal clinics in the Western Cape, South Africa. GBS were detected by culture and PCR and serotypes confirmed by latex agglutination tests. Antibiotic sensitivity was performed using disc diffusion.Results: The GBS colonization rate was 16.6%. Serotype distribution revealed serotype V as the predominant serotype (66.67%) followed by serotype III (21.05%). Serotypes Ia, II, IV, and IX constituted 1.75% each and 3 GBS isolates were non-typeable. Serotype V demonstrated resistance to most of the antibiotics tested, while serotype III demonstrated better susceptibility, except for tetracycline. No significant differences were observed for GBS colonization or serotype distribution according to HIV status.Conclusion: Predominating serotypes differed from those previously reported from other regions in South Africa. Global surveillance of serotype distribution plays an important role in informing vaccine development and antibiotic prophylaxis. Streptococci (GBS) or Streptococcus agalactiae, may exist as part of the normal microbiota in the female genital tract and anal areas of healthy adults, with the gastrointestinal tract serving as the natural reservoir and source of vaginal colonization , preterm rupture of membranes (PROM) and perinatal transmission of the organism . IntrautGBS infection in the newborn remains the foremost cause of neonatal morbidity and mortality globally, despite a recent decline in occurrence  with theIntrapartum colonization is strongly associated with early onset GBS sepsis, resulting in ~4% of reported fatalities and serious morbidities including sepsis, pneumonia, meningitis, illness, and death in infants, along with long-term disabilities . Other wThe etiology of LOD is not as well-understood as EOD and may involve community or nosocomial acquisition . HoweverIntrapartum antibiotics are known to reduce EOD in many countries , but in For the above reasons, the Centres for Disease Control and Prevention  recommenThis study included 301 women at 28–37 weeks of gestation, recruited from four antenatal clinics stationed in Mowbray, Mitchells Plain, Guguletu, and Khayelitsha in the Western Cape, South Africa. The participants were from lower socioeconomic groups, representative of two different ethnic groups, namely, black, and mixed-race groups. Patient details were captured in a brief questionnaire to which a sample number was assigned to ensure anonymity.Excluded from the study were women who reported bacterial infections for which antimicrobials were administered within 2 weeks prior to recruitment. Also excluded were those with any reported condition for which clinical vaginal sampling may be contraindicated.The content and objectives of the study were explained to the participants and their consent obtained by signature on the appropriate consent forms. Ethics approval for this study was obtained from the human ethics committee of the University of the Western Cape (UWC). The study complied with the declaration of Helsinki .A vaginal and anorectal swab were collected from each patient by trained nurses, using 2 separate sterile cotton-tipped swabs. Vaginal swabs were collected by inserting the swab about 3 cm into the vaginal wall and rotating the swab circumferentially. Anorectal swabs were collected by swabbing through the anal sphincter. Swabs were transported to the laboratory in carefully labeled liquid Amies with charcoal medium  and processed within 4 h of collection.Samples were inoculated in the order of first streaking onto enriched blood supplemented CNAgar  followed by inoculation in enriched broth (Todd-Hewitt Broth) and selective broth . The agar and broths were then incubated at 37°C for 24 h (h) in order to ensure optimal recovery of GBS. Media showing no growth after 24 h were incubated for a further 24 h before being declared as negative for growth. Culture purity was confirmed by subculture onto 5% sheep blood agar and microscopy using Kopeloff's modification of the Gram stain .Confirmation of GBS was achieved using catalase, Lansfield's grouping , esculin hydrolysis, and CAMP  tests.4)2 SO4, 20 mM MgSO4, 1% (w/v) Triton X-100), 0.2 mM each of dATP, dCTP, dGTP, and dTTP, 0.5 μM of each primer, 1U of Taq DNA polymerase, and 10 ng template DNA.DNA extraction was based on a procedure described by Sambrook et al.  and the After the initial heating step of 94°C for 4 min, cycles totalling 30 were run under the following conditions: denaturing at 94°C for 30 s, primer annealing at 50°C for 30 s and extension @ 72°C for 1 min 30 s and final extension at 72°C for 10 min. The universal bacterial 16S rRNA gene primer pair of E9F  and U151GBS specific primers (Sag 40 and Sag 445) were used for PCR amplification under the following conditions: A pre–PCR step at 94°C for 2 min, initial denaturation at 94°C for 45 s followed by 35 cycles of three temperature cycles involving denaturation at 95°C for 30 s, annealing at 60°C for 1 min, extension at 72°C for 2 min, followed by a final extension at 72°C for 10 min. The presence of each PCR product was then verified using electrophoresis in 1% agarose gel and stained with ethidium bromide. GBS template DNA was used as a positive control and no target DNA was added to the negative control.Streptococcus Group B typing kit, Statens Serum Institut, Denmark). A colony of GBS was suspended and incubated overnight in 5 ml of THB (Todd-Hewitt broth). Approximately 10 μl of the bacterial suspension was added to the latex suspension on the circle card and mixed by gentle shaking for a few seconds. Serotype designation was determined by a strong agglutination reaction within 5–10 s. Any agglutination after 30 s was not considered positive. If no designation could be made, the isolate was deemed non-typeable.GBS serotyping of the capsular polysaccharide antigens I-IX was achieved using a latex agglutination typing kit  for windows version 19.0. The Chi square test was used to determine the associations between two categorical variables, but when the number in a cell was <5, Fisher exact test was applied. A Lancefield grouping confirmed that 57 GBS isolates were obtained from the agar and broth cultures of 50/301 (16.6%) mothers. Comparison of agar and broth cultures showed no difference in isolation of GBS, while all of the isolates which were GBS culture-positive, were also positive by PCR. There was no significant difference between GBS isolated from the vagina (26/57) and the rectum (23/57) in this study. Twenty—three samples with negative vaginal cultures were positive by their corresponding anorectal samples, while GBS was isolated from 8 mothers by both vaginal and rectal samples.Universal bacterial primers E9F and U1510R showed that the DNA was suitable for PCR amplification and aided in determining the amount of metagenomic DNA to be used (5 ng) in all PCR experiments.The PCR product amplified with GBS-specific forward and reverse primers (Sag 40 and Sag 445) and viewed on a 1% agarose gel, yielded the expected band size of 405 bp.Although mothers from Mowbray and Khayelitsha showed similar rates of GBS colonization (36 and 32% respectively), significant differences in colonization rates were noted when Mitchells Plain (24%) and Guguletu (8%) mothers were included in the comparison (Table The mean age of the study population was 28.8 (±5.2) years and their ages ranged between 17 and 42 years (Table Most women 53.2%) reported their marital status as unmarried and 44.52% reported being married. No response to this question was received from the remaining 2.33% (Table % reporteThe majority of participants indicated that they had completed high school (91%) with <5% having primary school education and only 3.3% having had tertiary education Table . Three (The sample group comprised of mostly black (African) and mixed race (Colored) mothers, the majority of whom (59.8%) were unemployed Table . A largeHand washing of the body using a basin was the most frequently reported means of hygiene practice (68.7%), followed by bathing 24.1%), and shower (6.0%). Only 1 mother (0.3%) reported douching .HIV positivity (HIV+) was reported by 24.6% of the mothers, with 66.8% being HIV-negative (HIV–) and 8.6% either ignorant of their status or unwilling to disclose it. GBS colonization was higher in the HIV- mothers (62%) than in the HIV+ mothers (30%), (Table p > 0.05). One woman (HIV–) harbored more than 1 serotype of GBS, namely serotypes IV and V. No strains belonging to serotypes Ib, VI, VII, and VIII were found in any of the isolates in this study. Serotyping was not possible in 3 cases (5.3%), and were thus defined as non-typeable (NT). In eight cases where GBS was cultured from both rectal as well as vaginal samples, serotyping resulted in identical findings for all of the paired samples, except for three. The remaining three women were colonized with two different serotype combinations, namely, serotypes III/IX, serotypes IV/V, and serotypes III/V from the vaginal and anorectal swabs, respectively.Latex agglutination identified six different serotypes Figure . SerotypFifty-six of the fifty-seven GBS isolates (98%) were susceptible to clindamycin and erythromycin and 96% (55/57) were susceptible to penicillin G. Only 2% (1/57) showed susceptibility to tetracycline (T) and trimethoprim (TM) Figure . Total rExamination of the susceptibility profiles of the GBS serotypes are summarized in Table A report of invasive GBS disease in South Africa between 2004 and 2008 revealed an overall rate of neonatal GBS disease in the Western Cape as 0.53–0.67/1,000 live births . NeonataApartheid were classified as “Colored” (a term considered by many to be derogatory) and now fall under the generic “Black” category, which includes all race groups other than White. Because we were ethically bound to respect their refusal to be assigned to any particular race group against their will, we recorded them in the “missing” data, regardless of their appearance.In the present study, we followed the CDC guidelines  for sampLow socio-economic status may be associated with the level of education, access to preventive health care and poor personal and environmental hygiene. Many of the Black people included in this study came from informal settlements with undesirable housing and ablution facilities, which could account for the higher prevalence of GBS in this group, particularly since the majority reported hand basin body washing (which facilitates translocation of GBS) as their practice of personal hygiene. Although the location of the MOU (Midwife Obstetrics Unit) showed a significant association with GBS colonization, race (probably due to the uncategorised persons) did not. Also of note, is that the Mowbray MOU serves both race groups. Personal hygiene, employment, level of education, marital status, and age showed no significant association with GBS colonization.A prevalence rate similar to this study (16.5%) was reported from Germany  and 17.2Variations between different countries could be due to multiple factors including time of sampling , sampling site  and detection techniques (enriched selective vs. enriched media and /or PCR) as well as maternal socio-economic and other personal demography , 35. In Previous reports indicated that while GBS were isolated from only rectal specimens in some cases and from vaginal specimens in other cases, swabbing both the vagina and rectum was found to increase the yield substantially compared with sampling either the vagina or the rectum alone . Eight wp < 0.0001) and III (p = 0.027) in HIV+ compared to HIV– mother-neonate dyads, supporting the hypothesis of an increased risk for neonatal GBS infection with reduced protective antibodies  and III (p = 0.027) in HIV+ compared to HIV– mother-neonate dyads, suggesting an increased risk for neonatal GBS infection with reduced protective antibodies  were non-typeable in the present study, while only 0.7% of isolates were non-typeable in the Pretoria study . In the Just as different serotypes predominate in different population groups, so do their antimicrobial profiles. GBS susceptibility has frequently been reported to ampicillin, cefazolin, erythromycin, imipenem, norfloxacin, penicillin, and vancomycin . PenicilSerotypes III and V are reported as the most resistant serotypes with resistance to erythromycin, trimethroprim/sulphamethoxazole (TM/SMX), clindamycin and tetracycline reported , 55, 56.Strategies to prevent neonatal GBS infection include the elimination of exposure to GBS, chemoprophylaxis and vaccines. Intrapartum chemoprophylaxis has greatly reduced the risk of neonatal infection in women who carry GBS , but desAll the GBS strains isolated in the present study were resistant to fusidic acid, while others demonstrated resistance to TM/SMX (98%), tetracycline (98%), and gentamicin (93%). Resistance of GBS to tetracycline in our study population was similar to the results observed in other studies , 57. In A strength of this study is that to our knowledge it is the first to report on the antimicrobial resistance of GBS serotypes in the Western Cape as well as serotype differences in HIV+ vs. HIV– mothers. Limitations of the study include the relatively small sample number, reluctance of some participants to reveal their racial or HIV status, low income settings biased toward urban areas thereby eliminating a comparison with rural or advantaged socio-economic groups, and failure to follow up on neonates born of these mothers.GBS culture is labor intensive with the possibility of mixed cultures resulting in the overgrowth of competitive species and incubation time. Molecular diagnostic techniques such as PCR have the advantage of rapidly identifying bacteria which are difficult to grow and identifying newly emerging strains of bacteria. The benefit of rapid reporting of bacteria in pregnant women can significantly impact their pregnancy outcomes and reduce mortality rate, while allowing for a simpler and more efficient prevention programme which is speedy, sensitive, and precise. Thus, PCR offers a good alternative to culture in the absence of susceptibility testing, with 100% sensitivity and specificity reported in the present study when compared with culture.In conclusion, the results of our study differ considerably from studies in other regions of South Africa as well as in other parts of the world, with differences in serotype distribution and antimicrobial susceptibility possibly serving as a basis for GBS guidelines for South Africa. Our results stress the importance of epidemiological updates, particularly for those with penicillin allergies. More studies are needed to accurately estimate the global burden of GBS especially in developing countries . Improvein vitro laboratory investigations and preparation of the first draft of the manuscript. Both authors read and approved the final manuscript.CA designed the study, supervised the laboratory work and wrote the final paper. EK was responsible for the data collection, 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.