In fact, TAT, and particularly limb fat and SAT, but also VAT and trunk fat, all tended to increase regardless of the regimen, but only significantly so in those randomized to ATV/r. In the CASTLE study, a comparable increase in adipose tissue was observed 96 weeks after starting ritonavir-boosted

ATV [35]. A similar pattern was observed for lean body mass as well as total body mass changes. Early changes in body composition, after cART is first initiated, may at least partially reflect a restoration to normal health. Virological and immunological efficacy was similar in the two arms and therefore do not offer a likely explanation for the difference in body composition changes click here observed. The higher frequency of low-grade diarrhoea in the SQV/r arm may have contributed to the lower gain in lean body mass and adipose tissue. Another possible explanation is that, for six of the SQV/r-treated patients, but only one ATV/r-treated patient, only baseline and no follow-up DXA and CT scans were obtained. Given that missing values following baseline were imputed using a LOCF approach, this imbalance in available follow-up scans could have contributed to the apparent differences in fat gain learn more between the arms in the ITT analysis, which seems to be supported by the reduced difference observed in the OT analysis of adipose tissue changes. Detrimental effects of SQV on adipocyte differentiation and metabolism

have been reported [36,37]. Whereas ATV by itself has not been clearly demonstrated to affect adipocytes in vitro [38,39], another in vitro study showed that treatment with ritonavir-boosted ATV resulted in decreased adipocyte differentiation and insulin sensitivity, and

promoted oxidative stress and inflammation Ribose-5-phosphate isomerase [40]. TDF has been associated with nephrotoxicity, the risk of which may be increased by concomitant use of ritonavir-boosted PIs [24,41], potentially by increasing TDF exposure [25]. There is little information about whether this effect differs between PIs. The CASTLE study did not reveal a difference between ATV/r and lopinavir/r, combined with TDF, in the change in eGFR, with only a minor decrease in eGFR in both regimens [42]. The decline in eGFR observed in our study was also minor, developing during the first 12–24 weeks with no changes thereafter, as reported previously [24,41,42]. Only when estimated by CG was the decline in eGFR significantly greater for patients randomized to SQV/r. As the CG (but none of the other estimations) includes weight, the significantly greater increase in weight in ATV/r-treated patients could explain these findings, similar to the suggestions of others [43]. GFR estimated by weight-independent equations such as MDRD or CKD-EPI may offer a more reliable assessment of GFR after the initiation of first-line cART, a period which may be accompanied by significant weight change. Clinically relevant proximal tubulopathy was not observed with either treatment regimen.

In fact, TAT, and particularly limb fat and SAT, but also VAT and trunk fat, all tended to increase regardless of the regimen, but only significantly so in those randomized to ATV/r. In the CASTLE study, a comparable increase in adipose tissue was observed 96 weeks after starting ritonavir-boosted

ATV [35]. A similar pattern was observed for lean body mass as well as total body mass changes. Early changes in body composition, after cART is first initiated, may at least partially reflect a restoration to normal health. Virological and immunological efficacy was similar in the two arms and therefore do not offer a likely explanation for the difference in body composition changes click here observed. The higher frequency of low-grade diarrhoea in the SQV/r arm may have contributed to the lower gain in lean body mass and adipose tissue. Another possible explanation is that, for six of the SQV/r-treated patients, but only one ATV/r-treated patient, only baseline and no follow-up DXA and CT scans were obtained. Given that missing values following baseline were imputed using a LOCF approach, this imbalance in available follow-up scans could have contributed to the apparent differences in fat gain GSK2118436 concentration between the arms in the ITT analysis, which seems to be supported by the reduced difference observed in the OT analysis of adipose tissue changes. Detrimental effects of SQV on adipocyte differentiation and metabolism

have been reported [36,37]. Whereas ATV by itself has not been clearly demonstrated to affect adipocytes in vitro [38,39], another in vitro study showed that treatment with ritonavir-boosted ATV resulted in decreased adipocyte differentiation and insulin sensitivity, and

promoted oxidative stress and inflammation filipin [40]. TDF has been associated with nephrotoxicity, the risk of which may be increased by concomitant use of ritonavir-boosted PIs [24,41], potentially by increasing TDF exposure [25]. There is little information about whether this effect differs between PIs. The CASTLE study did not reveal a difference between ATV/r and lopinavir/r, combined with TDF, in the change in eGFR, with only a minor decrease in eGFR in both regimens [42]. The decline in eGFR observed in our study was also minor, developing during the first 12–24 weeks with no changes thereafter, as reported previously [24,41,42]. Only when estimated by CG was the decline in eGFR significantly greater for patients randomized to SQV/r. As the CG (but none of the other estimations) includes weight, the significantly greater increase in weight in ATV/r-treated patients could explain these findings, similar to the suggestions of others [43]. GFR estimated by weight-independent equations such as MDRD or CKD-EPI may offer a more reliable assessment of GFR after the initiation of first-line cART, a period which may be accompanied by significant weight change. Clinically relevant proximal tubulopathy was not observed with either treatment regimen.

All five SQ-degrading RGFP966 in vivo bacteria from Europe, including a strain of Pseudomonas putida, released sub-stoichiometric amounts of sulfate from SQ (Roy et al., 2000, 2003). Two organisms (e.g. Pseudomonas sp. and Klebsiella sp. strain ABR11) excreted organosulfonates (and, e.g. acetate), which were identified in the medium by C13-NMR as 3-sulfolactate and 2,3-dihydroxypropane-1-sulfonate (DHPS, sulfopropanediol) (Roy et al., 2003) (chemical structures in Fig. 1). Two organisms expressed phosphofructokinase, consistent with the operation of a glycolytic-type degradative pathway for SQ. Klebsiella sp. strain ABR11 also expressed an NAD+-dependent

SQ-dehydrogenase activity (Roy et al., 2003). More recently, organisms able to utilize sulfolactate and/or

DHPS have been discovered, and corresponding degradative pathways elucidated (e.g. Denger & Cook, 2010; Mayer et al., 2010). Further, sulfonate excretion systems in degradative pathways have been proposed (e.g. Weinitschke et al., 2007; Mayer & Cook, 2009; Krejčík et al., 2010). We wanted to use genome-sequenced organisms PI3K Inhibitor Library to expand on the work of Roy et al. (2000, 2003), but had little success with this approach, so we isolated an organism able to utilize SQ as a sole source of carbon and energy for growth. It was identified as a strain of P. putida, as found earlier by Roy et al. (2000), so we followed their lead to Klebsiella sp. and found that our sulfonate-utilizing Klebsiella oxytoca TauN1 (Styp von Rekowski et al., 2005) also utilized SQ. Each organism excreted a C3-sulfonate, which could be completely degraded by a second bacterium. Synthesis of SQ was Tryptophan synthase achieved following in part the protocols of Miyano &

Benson (1962) and of Roy & Hewlins (1997) without the need to form its barium salt for purification. The starting material for the preparation of SQ, 1,2-O-isopropylidene-6-O-tosyl-d-glucofuranose was prepared from 1,2-O-isopropylidene-d-glucofuranose by tosylation (Valverde et al., 1987) and isolated chromatographically pure. The tosylate (2.0 g) dissolved in ethanol (20 mL) was refluxed with an aqueous solution of Na2SO3 (1.21 g in 20 mL) under an inert gas atmosphere. Complete consumption of the starting tosyl compound (Rf: 0.62) was detected after 24 h by TLC in ethyl acetate on silica gel. Excess sodium sulfite was dissolved by the addition of water (50 mL) and the ethanol removed in vacuo. The aqueous solution was freed from sodium ions by passing it through a strongly acidic Amberlite IR 120 ion exchange column (45 g). Concentration of the acidic eluate under reduced pressure removed sulfur dioxide and cleaved the isopropylidene protecting group, leaving behind a syrup that consisted of equimolar amounts of p–toluenesulfonic acid and 6-sulfo-d-quinovose.

“
“Recently, a colleague Selleck GSK 3 inhibitor and I conducted a literature search concerning the stopping of medicines. Our search terms included ‘cessation’, ‘discontinuation’, ‘withdrawal’ and ‘stopping’, and we found some relevant studies, but not as many as we expected and felt that we must be missing something. We spoke to an Australian colleague who mentioned in passing the term ‘deprescribing’ which led us to

rerun our search with greater success. Although not in common parlance in the UK, as far I can discern, deprescribing was first used a decade ago in Australia by Woodward to describe the cessation of medicines.[1] Iyer et al. in their 2008 paper have described it as ‘medication withdrawal in older people’[2] and, more recently, it has been defined as ‘cessation of long-term therapy, supervised by a clinician’.[3] It is important to have a defined term to ensure shared understanding, and as advocated by Iyer et al.,[2] ‘deprescribing’ should be adopted internationally

by researchers and practitioners engaged in this area. There are many reasons why it may be desirable to withdraw a medicine from a patient: lack of efficacy, actual or potential adverse drug reactions, non-adherence, resolution of the condition, development of a contraindication, introduction of an interacting drug, to name a few. Most of click here the deprescribing literature focuses on older people; however, the above reasons

could apply to any patient. Nevertheless, there is a great deal of evidence of inappropriate prescribing in older people and they generally bear much of the burden of unnecessary polypharmacy with its associated morbidity. Coupled with the weak evidence-base for pharmacotherapy in older people, this population should be prioritised for deprescribing. Although the evidence-base for Cyclin-dependent kinase 3 deprescribing is limited due to mainly small, non-randomised or non-controlled studies, the weight of evidence shows that for most medicines included in the studies, deprescribing is not harmful in the majority of frail, older people and may be beneficial.[3, 4] For example, withdrawal of antipsychotics for challenging behaviour in dementia has been shown to reduce mortality in a randomised, placebo-controlled trial.[5] Garfinkel and Mangin, in a prospective cohort design, assessed the feasibility of the Good Palliative-Geriatric Practice algorithm in 70 older people over a mean follow-up period of 19 months and found that only 2% of 256 discontinued medicines needed to be restarted.[6] A similar previous study by the same authors in nursing home residents found that 10% of 332 medicines required restarting.[7] However, cessation of some medicines, particularly those affecting the central nervous system and the cardiovascular system, has the potential to cause adverse drug withdrawal events or recurrence of disease.

Single Sulfolobus colonies containing recombinant viral vectors were isolated by blue-white screening on rich media as described (Schleper et al., 1994). Virus infection was confirmed by PCR. Before all experiments, all strains containing viral vectors were grown to the stationary phase in minimal media containing 0.2% lactose and shifted to room temperature for 2 h to synchronize growth (Hjort & Bernander, 2001). Each culture was then diluted to OD600 nm=0.05

in yeast sucrose media, divided into three flasks, and incubated at 76 °C with moderate shaking. Cell-free extracts were prepared from 8.0 mL of OD600 nm=0.05 cultures 1 h after dilution for lag, 2.0 mL of OD600 nm=0.2 cultures for mid-exponential, and 0.3 mL of OD600 nm=1.2 cultures for stationary phase. Cultures were centrifuged for 10 min at 3000 g and cells were washed once in 1 sample volume of 10 mM Tris pH 8. Cells were resuspended in 400 μL 10 mM Obeticholic Acid Tris pH 8 and lysed by two freeze/thaw cycles of −80 and Adriamycin molecular weight 50 °C for 5 min each, and then diluted 1 : 10 in 10 mM Tris pH 8. Protein concentrations of cell-free extracts were determined by micro Bradford assay (Bio-Rad) compared with bovine serum albumin. β-Galactosidase

activities were determined by colorimetric endpoint enzyme assay (Jonuscheit et al., 2003). Briefly, 20 μL of each crude cell extract was added to 480 μL preheated 5 mM pNPG in 0.1 M sodium acetate pH 5. After 15 min at 95 °C (optimal temperature for lacS; Kaper et al., 2002), 1.0 mL of ice-cold 0.5M NaHCO3 was added and Protein Tyrosine Kinase inhibitor OD405 nm was measured spectrophotometrically. The amount of enzyme catalyzing the hydrolysis of 1 μmol of pNPG in 15 min at 95 °C is 1 U. The extinction coefficient of pNPG is 15.8 mM−1 cm−1 in sodium acetate pH 5 (Kaper et al., 2002). Extracts from S. solfataricus PH1 (lacS−) and S. solfataricus P1 (lacS wild type) served as negative and positive controls, respectively. Total DNA (Stedman et al., 1999) was

extracted from exponentially growing cultures (OD600 nm=0.2–0.3) of S. solfataricus PH1 infected with pMAD107 (16S/23S rRNAp-lacS), pMAD110 (TF55αp-lacS), or pKMSW72 (lacSp-lacS), digested with PstI, separated by gel electrophoresis, transferred and fixed to nitrocellulose membranes. Sulfolobus solfataricus PH1 chromosomal DNA and pKMSW72 plasmid DNA were included as size markers. The lacS gene was detected by a chemiluminescent probe complementary to the N-terminus of the gene and exposure to X-ray film (Supporting Information, Fig. S2). The vector copy number was determined from multiple exposures by comparing the intensity of the signals from the chromosomal and vector copies of lacS using imagequant (Molecular Dynamics). The absolute vector copy number in all cell-free extracts used for growth-phase dependent enzyme assays was determined by qPCR using the QuantiTect SYBR Green PCR kit (Qiagen) on a Strategene iCycler (Table S1). Vector-specific primers B49F and B49R were used at 0.

The patient was, therefore, admitted to our hospital for treatment, given intravenous infusions and observed for dengue warning signs. The patient’s platelet count was at its lowest on day 7 after onset

of disease (48 × 109/L) and her fever subsided on day 8 after onset. She was discharged after hospitalization for a total of 7 days. DENV-3 genome was detected by real-time polymerase chain reaction (RT-PCR, Applied Biosystems, USA) and virus isolated using the Aedes albopictus mosquito cell line C6/36.[3] Although tests for anti-dengue IgM (Focus Diagnostics, USA), and IgG (Panbio, Australia) antibodies were negative on day 2 after onset of disease, tests using serum sample from day 8 after http://www.selleckchem.com/products/ABT-263.html onset of disease was positive. Both day 2 and day 8 serum samples were positive for dengue NS1 antigen (Platelia, NVP-BKM120 Bio-Rad, France). Serum samples were de-identified prior to being used in the experiments and thus, ethical approval was not required for this study. The nucleotide sequence of the envelope protein (E-protein) of the isolated virus (GenBank accession number AB690858) was compared to selected sequences of DENV-3. The isolated DENV-3 strain from Benin belonged to DENV-3,

genotype III (Figure 1) and had the following characteristics: an E-protein sequence similarity of 99% to the DENV-3 D3/Hu/Côte d’Ivoire/NIID48/2008 strain, 99% to a DENV-3 strain isolated in Senegal in 2009, and 98% to a DENV-3 D3/Hu/Tanzania/NIID08/2010 strain isolated in Tanzania in 2010 (GenBank accession numbers: AB447989, GU189386, and AB549332, respectively). Sporadic cases or outbreaks of DENV infection have been reported in 34 countries in the African region. It is estimated Docetaxel chemical structure that 2.4% of global dengue hemorrhagic fever (DHF) cases (100,000 cases) and up to 1 million cases of DF may occur in Africa.[2] Among travel-associated dengue cases in travelers returning to Europe, 2 to 8% had visited Africa.[2, 5] In comparison, most of

the travelers returning to Europe with dengue had traveled to Asia (54–61%) and Latin America (25–31%). Febrile illness was, however, more frequently reported in 41% of travelers to sub-Saharan Africa (2,559 patients) as compared to other regions (Southeast Asia, 33%, 1,218 patients; Caribbean and Central and South America, 18%, 1,044 patients).[9] Although dengue is frequently reported in travelers to Southeast Asia and South America as compared to Africa, the disease may be underreported in Africa due to limited awareness of the disease, and, limited availability of diagnostic tests and routine surveillance system.[2] Imported cases of DENV type-3 infection from West Africa have been previously reported in European travelers.[2-6] The first possibility of DENV circulation in Benin was suggested by a seroprevalence study conducted in asymptomatic Germans working overseas from 1987 to 1993.

26; 95% CI 0.07–1.01). There was also a trend to lower HCV VL in this group, which may go some way to explaining this. Also, in a small French cohort of coinfected women (29% on HAART), rate of transmission did not

differ significantly between children born by vaginal delivery or CS [41]. HAART should be given to all HCV/HIV coinfected selleck screening library pregnant women, regardless of CD4 cell count or HIV VL because of the evidence of increased HIV transmission in coinfected mothers. 6.2.7 Where the CD4 cell count is <500 cells/μL, HAART should be continued if active HCV coinfection exists because of the increased risk of progressive HCV-related liver disease. Grading: 1B 6.2.8 Where the CD4 cell count is >500 cells/μL and there is no HCV viraemia or fibrosis, HAART should be discontinued. Grading: 2C 6.2.9 Where the CD4 cell count is >500 cells/μL and there is HCV viraemia and evidence of liver inflammation or fibrosis, continuing HAART is preferable because of a benefit on fibrosis progression.

GSK2126458 mouse Grading: 2B 6.2.10 Where the CD4 cell count is between 350 and 500 cells/μL and there is no evidence of viraemia, inflammation or fibrosis, continuing HAART is preferable if the patient displays a preference to do so. Grading: 2C The decision to continue ART or not postpartum depends on both HIV and HCV factors. There is consensus among guidelines that all persons with active (HCV-viraemic) coinfection should receive HAART if their CD4 cell count is <500 cells/μL [[2],[42],[43]]. In those women with CD4 cell counts of 350–500 cells/μL who have cleared infection either spontaneously (about 25%) or after treatment and with a sustained virological response (SVR) and who have normal liver histology as judged by biopsy or Fibroscan,

consideration should be given to continuing cART where the patient expresses a preference to do so. This is because until completion of the randomized PROMISE trial, which addresses the question of whether to continue HAART postnatally in mothers with CD4 cell counts >400 cells/μL, there is equipoise as to correct management. In those with CD4 cell counts >500 cells/μL, who received Florfenicol HAART to prevent MTCT, and who are not HCV-viraemic and have no evidence of established liver disease, ARVs can be discontinued. Without additional risk factors (such as alcohol, steatosis) and assuming they are not reinfected, these women should have no further histological progression of their liver. In women with CD4 cell counts >500 cells/μL who have established liver disease (inflammation or fibrosis), therapy should be continued. Interruption of ART in the SMART study was shown to lead to a greater risk of non-opportunistic disease-related death, particularly among those with HIV/HCV coinfection.

The response rate was 37.5% (150 questionnaires returned completed and suitable for analysis). The number of completed questionnaires obtained from each department is presented in Table 3. The distribution of participating PCPs was similar to the distribution of PCPs in Franche-Comté Dabrafenib cost (data from the Regional Heath Agency: Agence régionale de la santé ARS). The sociodemographic details and practice-related characteristics of the participating

PCPs are presented in Table 1. Only 50 PCPs heeded our request to choose only three pieces of priority health advice from the items proposed by the MCQ. The others selected all the items that seemed relevant in their opinion. Percentages of responses for each item are presented in Table 2. The three pieces of priority advice that should have been chosen were water hygiene recommendations (85%), use of antimosquito protection (70%), (advice on wearing long clothes in the evening was also accepted because of the possible contraindications of insect repellent during pregnancy, 55%), and the advice to cancel the

trip (25%). Most PCPs selected these items, except for cancelation of the trip. An expert opinion would have been requested by 17% of PCPs. The diphtheria–tetanus–poliomyelitis vaccine is the only jab that can be prescribed during pregnancy (59%). Safety of the hepatitis A vaccine (32%) was considered debatable. Hepatitis B (28%), yellow fever (25%), typhoid (18%), rabies (3%), meningitis (6%), and flu (5%) vaccines were considered inappropriate. Japanese encephalitis (0%), measles–mumps–rubella (6%), and tuberculosis find more (3%) vaccines were considered as incorrect answers (because they should be avoided during pregnancy). Twenty-five percent of PCPs selected the “no vaccination” item. An expert opinion would have been requested by 43% of PCPs. Appropriate malaria chemoprophylaxis was mefloquine (13%) or atovaquone + proguanil (24%).

ADAMTS5 Inappropriate protection would have been prescribed by 16% of PCPs, with 7% prescribing chloroquine and 9% chloroquine + proguanil. Thirty-one percent of PCPs chose not to use chemoprophylaxis in spite of the seriousness of malaria infection during pregnancy, and 3% of PCPs would prescribe doxycycline even though this treatment is to be avoided during pregnancy. An expert opinion would have been requested by 44% of PCPs. The three pieces of priority advice that should have been chosen were water hygiene recommendations (88%), hand hygiene recommendations (66%), and the use of antimosquito protection (77%), especially because the patient’s trip was planned during the wet season. PCPs mostly answered correctly and they also often selected the “repatriation insurance” item (66%), probably due to the age and diabetic condition of the patient. An expert opinion would have been requested by 17% of PCPs.

A paired-pulse transcranial magnetic stimulation paradigm was used in order to evaluate and compare the PMv–M1 interactions during different phases (rest, preparation and execution) of an index finger movement in patients with FHD and controls. A sub-threshold conditioning pulse (80% resting motor threshold) was applied

to the PMv at 6 ms before M1 stimulation. The right abductor pollicis brevis, a surround OSI-906 clinical trial muscle, was the target muscle. In healthy controls, the results showed that PMv stimulation induced an ipsilateral ventral premotor–motor inhibition at rest. This cortico-cortical interaction changed into an early facilitation (100 ms before movement onset) and turned back to inhibition 50 ms later. In patients with FHD, this PMv–M1 interaction and its modulation were absent. Our results show that, although the ipsilateral ventral premotor–motor inhibition does not play a key role EPZ015666 in the genesis of surround inhibition,

PMv has a dynamic influence on M1 excitability during the early steps of motor execution. The impaired cortico-cortical interactions observed in patients with FHD might contribute, at least in part, to the abnormal motor command. A major feature of the pathophysiology of focal hand dystonia (FHD) is the lack of inhibition at the cortical, sub-cortical, and spinal levels, which is probably due to GABAergic dysfunction (Hallett, 2011). Impairment of intracortical circuits has been demonstrated in FHD, and this may be either an intrinsic abnormality or secondary to striatal dysfunction (Peller et al., 2006). In particular, surround inhibition (SI), which represents the suppression of excitability in the area surrounding an activated neural network in order to focus and select neuronal responses 3-oxoacyl-(acyl-carrier-protein) reductase (Sohn & Hallett, 2004b), is impaired in FHD (Sohn & Hallett, 2004a). The lack of SI might explain, at least in part, the excessive antagonist and accessory muscle activation

in patients with FHD (van der Kamp et al., 1989). The mechanisms responsible for SI are still unknown. No intracortical inhibitory circuit located in or projecting to the primary motor cortex (M1) has been identified as a source of SI (Beck & Hallett, 2011). As it starts during movement preparation, SI could result from connections between the M1 and premotor areas involved in hand motor control. Accordingly, Beck and colleagues investigated the potential role of the dorsal premotor cortex in the generation of SI. Indeed, the dorsal premotor cortex plays an important role in movement selection (Rushworth et al., 2003) and some imaging studies have shown an impairment of dorsal premotor cortex activation in right-sided FHD (Ceballos-Baumann et al., 1997; Ceballos-Baumann & Brooks, 1998; Ibanez et al., 1999). However, the results demonstrated that the ipsilateral dorsal premotor–motor inhibition was not involved in the genesis of SI (Beck et al., 2009a). The ventral premotor cortex (PMv) plays a key role in fine finger and hand movements.

cereus using this identification method, and the full sequence of the novel vip1 gene was obtained by single oligonucleotide nested (SON)-PCR. The novel vip1 and vip2 binary

toxin genes were co-expressed in the vector pCOLADuet-1, and their expression proteins were assayed against several insects. A type strain of B. cereus strain (CGMCC ID: 0984) was obtained from China General Microbiological Culture Collection Center (CGMCC, Beijing, China). Twenty-five B. cereus strains were isolated from soils of Sichuan province, China. Bacillus cereus strain HL12 containing novel Vip1–Vip2 binary toxin was deposited in CGMCC (ID: 3921). The vector pCOLADuet-1 (Merck, Shanghai, China), containing two multiple cloning sites, was used to co-express vip1Ac1 and vip2Ae3 genes in Escherichia coli strain BL21 (Tiangen, Beijing, China). The genes were cloned into pMD19-t vector (TaKaRa, Birinapant supplier Japan) and transformed into E. coli strain DH5α (Tiangen) for nucleotide sequencing. The Vip1s and Vip1a primers (Table 1) were designed based on the conserved region for characterization of the

IDH inhibitor cancer vip1 genes (Yu et al., 2010). The length of PCR product was about 500 bp. Another primers set, Vip1f and Vip1r (Table 1), was designed to amplify a 1140-bp DNA fragment for the PCR–RFLP assay. These primers were designed by aligning the vip1-subgroup gene (vip1Aa3, vip1Ba2, vip1Ca1, and vip1Da1) sequences with GenBank accession numbers of GU992203, AJ872073, AY245547, and AJ871923. All of the primers used in this study are shown in Table 1. PCR amplification was performed as follow: 95 °C for 5 min (initial denaturation), 34 cycles at 95 °C for 1 min, annealing temperature (Table 1) for 1 min, and 72 °C for extension for 1 min, followed by a final extension at 72 °C for 7 min. To determine the bacterial strains that contained vip1 genes, PCR was performed with Vip1s and Vip1a primer pair. Strains with Metalloexopeptidase the vip1 genes were selected to perform PCR amplification with the Vip1f and Vip1r primer set, and the PCR amplicons were purified from agarose gel using the AxyPrep DNA Gel extraction kit (Ayxgen Biosciences). Nucleotide

sequences of vip1Aa3, vip1Ba2, vip1Ca1, and vip1Da1 were used as references to identify suitable endonucleases in silico. Restriction analysis simulation using MapDraw5.0 (DNAStar) identified the AciI as an effective endonuclease with high discriminatory potential, so AciI was used to digest the recovered PCR amplicons. The expected restriction fragment size of the reference vip1-type genes is shown in Table 2. The restriction analysis was carried out in a total volume of 20 μL consisting of 2 μL of 10× digestion buffer (100 mM NaCl, 50 mM Tri–HCl, 10 mM MgCl2, 1 mM DTT, pH 7.9), 1 μL of AciI (New England Biolabs, Beijing, China) endonuclease, 1 μL PCR product (about 1 μg mL−1), and 16 μL deionized water. All digestions were carried out at 37 °C for 3 h, and the digested products were separated by electrophoresis in 1.5% agarose gel.