Our results demonstrate that cells coexpressing doublecortin and PSA-NCAM, but lacking neuronal nuclear antigen expression, were present in the amygdala of adult humans. These cells were organised in elongated

clusters, which were located between the white matter of the dorsal hippocampus and the basolateral amygdaloid nucleus. These clusters were not associated with astroglial specialised structures. No cells expressing the proliferative marker Ki67 were observed in the amygdaloid parenchyma, although some of them were found in the vicinity of the lateral ventricle. Immature neurons were also present in the amygdala of squirrel monkeys and cats. These cells also appeared clustered in monkeys, although not as organised as in humans. In cats these cells are scarce, appear isolated and most of the PSA-NCAM-expressing structures LY2835219 clinical trial corresponded to processes apparently originating from the paleocortical layer II. “
“Callous-unemotional violence associated with antisocial personality disorder is often

find more called ‘predatory’ because it involves restricted intention signaling and low emotional/physiological arousal, including decreased glucocorticoid production. This epithet may be a mere metaphor, but may also cover a structural similarity at the level of the hypothalamus where the control of affective and predatory aggression diverges. We investigated this hypothesis in a laboratory model where glucocorticoid production is chronically limited by adrenalectomy with glucocorticoid replacement (ADXr). This procedure was proposed to model important aspects of antisocial violence. Sham and ADXr rats were submitted to resident/intruder conflicts, and the resulting neuronal activation patterns were investigated by c-Fos immunocytochemistry.

In line with earlier findings, the share of attacks aimed at vulnerable targets (head, throat and belly) was dramatically increased by ADXr, while intention signaling by offensive threats was restricted. Aggressive encounters activated the mediobasal hypothalamus, a region involved in intra-specific aggression, but sham and ADXr rats did not differ in this respect. In contrast, the activation of the lateral hypothalamus that is tightly involved in predatory aggression was markedly larger in ADXr cAMP rats; moreover, c-Fos counts correlated positively with the share of vulnerable attacks and negatively with social signaling. Glucocorticoid deficiency increased c-Fos activation in the central amygdala, a region also involved in predatory aggression. In addition, activation patterns in the periaqueductal gray – involved in autonomic control – also resembled those seen in predatory aggression. These findings suggest that antisocial and predatory aggression are not only similar but are controlled by overlapping neural mechanisms. “
“Nicotine, a major psychoactive component of tobacco smoke, increases glutamate transmission in the nucleus accumbens (NAcc).

1), streptococcal culture FDA-approved Drug Library ic50 supernatants were harvested

by centrifugation exactly at the mid log phase (A600 = 0.6–0.7) (Svensson et al., 2002). In addition, to ensure the absence of the zymogene (40 kDa) and active form (28 kDa) of the SpeB protease, the proteins of mid log phase culture supernatants were precipitated using 100% trichloroacetic acid and were evaluated by standard 12% SDS-PAGE and Coomassie blue staining (Svensson et al., 2002). The culture supernatants were subsequently filtered through 0.22-μm filter (Whatman, Germany) and stored at −70 °C until use. For each colorimetric assay, 50 μL of the culture supernatant was incubated at 37 °C with 100 μL 50 mM Tris–HCl, pH 7.4 in microplate containing 50 μg mL−1 of human Plg (Sigma) for 15 min. The S-2251 substrate (50 μL of 2.5 mM) was added, and absorbance was measured at 405 nm every 5 min for 60 min. Each assay was performed in duplicate, both in the presence and absence of Plg/S-2251. Assays containing the intact (unused) THB media and culture supernatants of the reference strains were also employed as negative and positive controls, respectively. Serial dilutions of Streptase® (CSL, Behring, Germany), a commercial SK, were used to prepare the standard curve for

SK Selleck Ibrutinib activity. Optical densities were plotted against time, and activity rates were determined from linear portion of the curve. The level of SK activity in each bacterial culture supernatant was converted to IU mL−1 using the standard curve. The PCR product of a representative of each digestion pattern (Fig. 1) was selected for nucleotide sequencing. DNA sequencing data were used for alignment studies and restriction site mapping via application Nintedanib (BIBF 1120) of the Molecular Evolutionary Genetic Analysis (mega 4) analytical package (Tamura et al., 2007). Difference in SK activities among GAS and GCS/GGS groups was determined using the one-way

anova test. The Kruskal–Wallis analysis of variance was employed to calculate the level of significance of SK activity among variants. All statistical analyses were carried out using spss version 16.0 (SPSS, Inc., Chicago, IL). A value of P < 0.05 was considered significant. PCR-based amplification of the sk-V1 region produced the expected 339-bp fragments (Fig. 1). Digestion of the PCR products (sk-V1) by MluI, PvuII, DraI and DdeI restriction enzymes also provided exactly the earlier described restriction patterns of the DNA fragments (Tewodros et al., 1996) (Fig. 1, Supporting Information, Table S1). The reference strains GCS/GGS and NZ131 were classified as sk5 and sk1, respectively, as expected. In total, 21 sk allelic variants were detected among strains investigated in our study (Fig. 2). Besides the several previously reported sk allelic variants of GAS and GCS/GGS isolates (Tewodros et al.

However, approximately one-third (31.7%; CI 26.0–39.6%) did not expect pharmacists to be available for consultation during rounds. Physicians’ experiences with pharmacists were

less favourable; whereas 77% (CI 70.2–81.5%) of the physicians agreed that pharmacists were always a reliable source of information, only 11.5% (CI 6.2–16.4%) agreed that pharmacists appeared to be willing to take responsibility for solving any drug-related problems. The present study showed that hospital physicians are more likely to accept traditional pharmacy services than newer clinical services for hospital-based pharmacists in the West Bank, Palestine. Pharmacists should therefore interact more positively and more frequently with physicians. This will close the gap between the

physicians’ commonly held perceptions of what they expect pharmacists to do and ICG-001 what pharmacists can actually do, and gain support for an extended role of hospital-based pharmacists in future patient therapy management. “
“Feasibility of pharmacist delivered motivational interviewing (MI) to methadone patients has been demonstrated, but its efficacy is untested. This study aimed to determine whether pharmacists trained in MI techniques can improve methadone outcomes. A cluster randomised controlled trial by pharmacy, with community pharmacies across Scotland providing supervised methadone to >10 daily patients, aged >18 years, started on methadone <24 months. Pharmacies were randomised to intervention or control. Intervention pharmacists received MI training and a resource pack. Opaganib concentration Control pharmacists continued with normal practice. Primary outcome was illicit heroin use. Secondary outcomes were treatment retention, substance use, injecting behaviour, psychological/physical health, treatment satisfaction and patient feedback. Data were collected via structured interviews at baseline

and 6 months. Seventy-six pharmacies recruited 542 patients (295 intervention, 247 control), mean age 32 years; 64% male; 91% unemployed; mean treatment length 9 months. No significant difference in outcomes between groups for illicit heroin use (32.4% cf. 31.4%), although within-groups use reduced (P < 0.001); treatment retention was Fenbendazole higher in the intervention group but not significantly (88% cf. 81%; P = 0.34); no significant difference between groups in treatment satisfaction, although this improved significantly in intervention (P < 0.05). More intervention than control patients said pharmacists had ‘spoken more,’ which approached statistical significance (P = 0.06), and more intervention patients found this useful (P < 0.05). Limited intervention delivery may have reduced study power. The intervention did not significantly reduce heroin use, but there are indications of positive benefits from increased communication and treatment satisfaction. Methadone is the most commonly prescribed opiate replacement treatment in Scotland.

, 1994). We cannot exclude that in contrast to pri2 in S. commune,

priB does play a role in mushroom formation in L. edodes. The monokaryotic Δjmj3 strain was also indistinguishable from the wild-type strain. However, the dikaryotic Δjmj3Δjmj3 strain grew somewhat slower than the wild type and formed a less dense mycelium. Yet, the mutant did form sporulating fruiting bodies (data not shown). Taken together, we have shown that the relative incidence of gene inactivation by homologous integration is drastically increased in a Δku80 strain when compared with the wild type. This strain will therefore be highly find more instrumental in the functional analysis of genes in S. commune and, in this way, contribute towards our understanding of the biology of mushroom-forming basidiomycetes. This research was supported by the Dutch Technology Foundation STW, the Applied Science division of NWO and the Technology Program of the Ministry of Economic Affairs. “
“It is expected that Mannheimia hemolyticaA1 expresses a particular collection of genes during infection in the host. The bacterial gene products are produced in the in vivo environment to facilitate growth and survival. Here, we examined gene expression

by M. hemolyticaA1 in the bovine host after 6 days of infection. Total RNA from M. hemolyticaA1 recovered from pneumonic lungs of two animals was used to produce Temozolomide cDNA to screen a custom M. hemolyticaA1 microarray. The expression profile was compared to a RNA sample from an in vitro grown culture. The data showed that 44 genes were differentially expressed by more than eightfold when compared with the in vitro sample. Seventeen genes were found Niclosamide to have higher expression in vivo and 27 genes had lower expression. Several virulence-associated genes including those encoding leukotoxin, a capsule biosynthetic enzyme and the serotype-specific antigen, Ssa, had reduced expression, suggesting that their products may not be important during the later stages of infection. Most of the genes up-regulated in vivo encoded hypothetical or conserved hypothetical

proteins. Three Mu-like bacteriophage-related genes were up-regulated in the in vivo sample, suggesting that the prophage may be transcriptionally active. The results provide a glimpse of gene expression by the bacterium in the host after pulmonary infection has been established. Bovine pneumonic pasteurellosis is the major cause of morbidity and mortality in beef cattle in North America and results in significant economic loss to the cattle industry (Griffin, 1997). The primary causative agent of pneumonic pasteurellosis is Mannheimia hemolytica A1, a Gram-negative, non-motile bacterium that is a part of the normal flora of the upper respiratory mucosa of cattle (Frank, 1988). To-date, there is no information pertaining to the global gene expression of M. hemolytica A1 within the bovine host during an infection. Roehrig et al. (2007) examined the transcriptome profile of M.

, 2010). However, the regulation of other genes including ferBA remains unknown. While there are a number of reports on the catabolic genes of ferulate (Rahouti et al., 1989; Gasson et al., 1998; Venturi et al., 1998; Overhage et al., 1999; Achterholt et al., 2000; Civolani et al., 2000; Masai et al., 2002), the information regarding the transcriptional regulation of the ferulate catabolic genes is very scarce. However, Calisti et al. (2008) reported on regulation of the ferulate catabolic genes of Pseudomonas

fluorescens BF13. In this strain, the ech-vdh-fcs genes, which respectively encode PF-01367338 cost feruloyl-CoA hydratase/lyase, vanillin dehydrogenase, and feruloyl-CoA synthetase, formed an operon, and the transcription of this operon was negatively regulated by a MarR-type transcriptional regulator, FerR. Based on the ech promoter assay using BF13 mutants, feruloyl-CoA was identified as an inducer molecule of the ech-vdh-fcs operon. Similar this website observation had been described in the regulation of the hydroxycinnamate catabolic genes (hca) of Acinetobacter baylyi ADP1 (Parke & Ornston, 2003). The hca genes were shown to be negatively regulated by a MarR-type transcriptional regulator,

HcaR. Furthermore, p-coumaroyl-CoA was identified as a true inducer. However, the biochemical analysis of the interaction of the regulator protein with the operator sequence has not been documented, and there 17-DMAG (Alvespimycin) HCl is no direct proof that hydroxycinnamoyl-CoAs are the effector molecules of these MarR-type transcriptional regulator proteins. In this study, we characterized the transcriptional regulation of the ferBA genes of SYK-6 controlled by

a MarR-type transcriptional regulator, FerC. In vitro assay demonstrated the interaction between FerC and the operator sequence, and actual effector molecules of FerC were identified. The bacterial strains and plasmids used in this study are listed in Supporting Information, Table S1. Sphingobium sp. strain SYK-6 and its mutant derivatives were routinely grown at 30 °C in Luria-Bertani (LB) medium or Wx minimal salt medium (Table S2) containing 5 mM ferulate, 5 mM vanillin, 10 mM vanillate, or SEMP (10 mM sucrose, 10 mM glutamate, 0.34 mM methionine, and 10 mM proline). The ferC mutant, SME043 was obtained by introduction of the ferC disruption plasmid, pFESIBI into the cells of SYK-6, and disruption of the gene was examined by Southern hybridization analysis as described previously (Sato et al., 2009). Escherichia coli strains were grown in LB medium at 37 °C. For cultures of cells carrying antibiotic resistance markers, the media for E. coli transformants were supplemented with 100 mg of ampicillin per liter or 25 mg of kanamycin per liter.

Using both in-vivo recordings combined with microiontophoretic or intraperitoneal drug applications and in-vitro experiments, we have found that M-type channels, which are present in midbrain dopaminergic cells, Staurosporine mouse modulate the firing during bursting without affecting the background low-frequency pacemaker firing. Thus, a selective blocker of these channels, 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride, specifically potentiated burst firing.

Computer modeling of the dopamine neuron confirmed the possibility of a differential influence of M-type channels on excitability during various firing patterns. Therefore, these channels may provide a novel target for the treatment of dopamine-related diseases, including Parkinson’s disease and drug addiction. Moreover, our results demonstrate that the influence of M-type channels on the excitability of these slow pacemaker neurons is conditional upon their firing pattern. “
“The mouse cerebellum consists of 10 lobules, which are distinguishable by their anatomical and functional properties. However, the differences in the slow postsynaptic currents (sPSCs) of Purkinje cells between lobules have not been well studied. We recorded the sPSCs of lobules 3, 9 and 10 evoked by tetanic stimulation of the molecular layer in cerebellar slices, Protein Tyrosine Kinase inhibitor and found

a novel outward sPSC mediated by the GABAB receptor in loblues 9 and 10 but hardly at all in lobule 3. We showed that the lobule-specific difference is at least partly attributable to differences in the density of

GABAergic neurons (higher in lobule 10 than in lobules 3 and 9), and the functional expression level of postsynaptic GABAB receptor currents (larger in lobules 9 and 10 than in lobule 3). The G-protein-coupled inward rectifying K+ channel (GIRK) is known to be activated by GABAB receptors; however, the outward sPSC was not blocked by a GIRK blocker, was not sensitive to Cs+ Palmatine block, and was observed when Cs+ was used as a charge carrier. These results suggest that a K+ channel other than GIRK could be activated by GABAB receptors. KCNK13 is a Cs+-permeable K+ channel that shows intense expression of mRNA in Purkinje cells. KCNK13 current was enhanced by co-expression of Gβγ subunits and was observed when Cs+ was used as a charge carrier in heterologous expression systems, and the amino acids critical for these features were identified by mutagenesis. Taken together, these results show that KCNK13 is a legitimate candidate for the Cs+-permeable K+ channel activated by GABAB receptors, presumably via Gβγ subunits in Purkinje cells. “
“Division of Translational Research for Drug Discovery, Fukushima Medical University, Fukushima, Japan Cathepsin C (CC) (EC 3.4.14.1, dipeptidyl peptidase I) is a lysosomal cysteine protease that is required for the activation of several granule-associated serine proteases in vivo.

Using both in-vivo recordings combined with microiontophoretic or intraperitoneal drug applications and in-vitro experiments, we have found that M-type channels, which are present in midbrain dopaminergic cells, Trametinib research buy modulate the firing during bursting without affecting the background low-frequency pacemaker firing. Thus, a selective blocker of these channels, 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride, specifically potentiated burst firing.

Computer modeling of the dopamine neuron confirmed the possibility of a differential influence of M-type channels on excitability during various firing patterns. Therefore, these channels may provide a novel target for the treatment of dopamine-related diseases, including Parkinson’s disease and drug addiction. Moreover, our results demonstrate that the influence of M-type channels on the excitability of these slow pacemaker neurons is conditional upon their firing pattern. “
“The mouse cerebellum consists of 10 lobules, which are distinguishable by their anatomical and functional properties. However, the differences in the slow postsynaptic currents (sPSCs) of Purkinje cells between lobules have not been well studied. We recorded the sPSCs of lobules 3, 9 and 10 evoked by tetanic stimulation of the molecular layer in cerebellar slices, Epacadostat clinical trial and found

a novel outward sPSC mediated by the GABAB receptor in loblues 9 and 10 but hardly at all in lobule 3. We showed that the lobule-specific difference is at least partly attributable to differences in the density of

GABAergic neurons (higher in lobule 10 than in lobules 3 and 9), and the functional expression level of postsynaptic GABAB receptor currents (larger in lobules 9 and 10 than in lobule 3). The G-protein-coupled inward rectifying K+ channel (GIRK) is known to be activated by GABAB receptors; however, the outward sPSC was not blocked by a GIRK blocker, was not sensitive to Cs+ selleck inhibitor block, and was observed when Cs+ was used as a charge carrier. These results suggest that a K+ channel other than GIRK could be activated by GABAB receptors. KCNK13 is a Cs+-permeable K+ channel that shows intense expression of mRNA in Purkinje cells. KCNK13 current was enhanced by co-expression of Gβγ subunits and was observed when Cs+ was used as a charge carrier in heterologous expression systems, and the amino acids critical for these features were identified by mutagenesis. Taken together, these results show that KCNK13 is a legitimate candidate for the Cs+-permeable K+ channel activated by GABAB receptors, presumably via Gβγ subunits in Purkinje cells. “
“Division of Translational Research for Drug Discovery, Fukushima Medical University, Fukushima, Japan Cathepsin C (CC) (EC 3.4.14.1, dipeptidyl peptidase I) is a lysosomal cysteine protease that is required for the activation of several granule-associated serine proteases in vivo.

pylori isolates. Notably, peptidyl-prolyl cis–trans isomerase (PPIase) was detected positively in 11 out of 22 (50%) gastric cancer-associated H. pylori strains. In contrast, <24% of the H. pylori strains from superficial gastritis showed positive results. Given the potential role of PPIases in cell growth, apoptosis and oncogenic transformation, our results suggest that PPIase may represent a novel marker and potential therapeutic target for gastric cancer. Helicobacter pylori is a microaerophilic Gram-negative

bacillus that colonizes the stomach in more than half of the world’s population (Parsonnet, 1995). It is the causative agent of chronic gastritis and contributes to peptic ulcer. There is strong evidence Small molecule library concentration to indicate that H. pylori plays an important role in the pathogenesis of noncardia gastric cancer Group HaCC (2001). Once infected by this bacterium, the clinical outcome depends on the interaction of virulent effects of the bacterium, the host response and the environment. DNA fingerprinting studies revealed considerable diversity among independent H. pylori isolates (Akopyanz et al., 1992). This observation was supported by later studies using multilocus enzyme electrophoresis analysis (Go et al., 1996) and restriction fragment length polymorphisms analysis click here (Salaun et al., 1998). Genes that are present in one

strain and absent or substantially different in the others can be of significant biological interest. The CagA protein was one of the first several virulent determinants and disease markers identified in H. pylori. Previous studies have demonstrated that strains lacking the cagA gene, which are common in Europe and North America, are rarely implicated in overt disease (Mimuro et al., 2008). Patients with high titers of anti-CagA antibodies tend to have a higher risk of developing peptic ulcer or gastric cancer (Covacci et al., 1993). The genetic heterogeneity of H. pylori is believed to be geographical and may occur via DNA rearrangement and the introduction and deletion of foreign sequences (Achtman & Suerbaum, 2000). In

contrast to H. pylori strains from Western countries, most East Asian strains express CagA (Yamaoka et al., 2008). Furthermore, heterogeneity within CagA exists between strains from Western and East Asian countries (Yamaoka Methocarbamol et al., 2000). The number of EPIYA-C repeat motifs in the C-terminus of CagA may be related to high incidences of gastric cancer, and thus, is proposed as a marker for clinical outcomes (Yamaoka et al., 1998). In an attempt to identify gastric cancer-specific H. pylori genes, we isolated H. pylori from both gastric cancer patients and superficial gastritis patients, and constructed a gastric cancer-specific gene library using a well-established suppression subtractive hybridization (SSH) method to selectively amplify target DNA fragments and simultaneously suppress nontarget DNA amplification (Diatchenko et al., 1996; Akopyants et al., 1998).

The HGM-3 model contains some nonroutine tests that are not widely available

and may be expensive if they were to perform the ELISA classic, which makes the model less attractive as it may not be possible for most clinicians to use it. However, at present, these molecules can be measured using a new Luminex-based assay in a quick and inexpensive way. HGM-3 also gave good results for cirrhosis diagnosis, but we found similar AUC-ROC values for HGM-3 and HGM-2. In our opinion, HGM-3 is less useful for diagnosis of cirrhosis or advanced fibrosis because HGM-2 is calculated from indirect markers associated with fibrosis such as routine biochemistry and platelet data which are widely available and very inexpensive [21]. Moreover, we were unable to assess the

diagnostic accuracy in the estimation and validation groups because of the low number of patients with cirrhosis included in this study. The identification Smad inhibitor of this index in HIV-positive individuals is also of importance as HIV infection may alter the expression of many of the immune, apoptotic and ECM markers. However, this study had several limitations. (1) The low number of patients. (2) The study was limited to patients with well-preserved immune function and extrapolation to individuals with more marked immune suppression will require further study. (3) We did not compare HGM-3 with SHASTA, Fibrotest, Hepascore and Fibrometer because we did not have all the variables needed to calculate these indexes. (4) HGM-3 was derived from the majority of this combined cohort and so would be expected to perform optimally in this cohort; HM781-36B chemical structure whereas the other indexes tested (APRI, FIB-4 and Forns’ indexes) were not optimized in this cohort and would be expected to perform less well. (5)

We cannot give exact information regarding biopsy length or portal tracts; however, we found that only 1.68% of biopsies Benzatropine were defective for pathological diagnosis and these cases were excluded from the study. In any case, the pathologist had samples of acceptable quality to make a diagnosis of fibrosis for 98.32% of obtained biopsies. In summary, we found that platelet count, ALP, HGF, TIMP-1 and HA were independent predictive markers of advanced fibrosis in HIV/HCV-coinfected patients. The combination of these indirect markers with direct markers of fibrosis in a logistic probability function yielded a new serum index that accurately predicted bridging fibrosis and cirrhosis. However, as with most models, HGM-3 better predicts the absence of fibrosis (97% certainty for F<3 fibrosis) than the presence of significant fibrosis (77% certainty). HGM-3 improves upon the accuracy of other previously published indexes but still has limitations in accurately identifying patients with F≥3. This indicates that further research should be carried out to improve the ability to diagnose advanced fibrosis (F≥3) in HIV/HCV-coinfected patients.

This step-by-step approach has helped women to gradually make difficult personal changes to their birth plans. The input of the MDT is crucial to support these women, as they are often the most isolated and unsupported. Where, despite all efforts, the MDT is unable to influence a mother’s views antenatally, a pre-birth planning meeting with social services should be held.

The mother should be informed that it is the paediatrician’s role to advocate on behalf of the child’s well-being and therefore to prevent, where possible, HIV infection. If the mother continues to refuse any intervention package, then legal permission should be sought at birth to treat the infant for 4 weeks with combination PEP and prevent breastfeeding. Preparation of the legal case may be lengthy and time consuming; useful documentation click here can be obtained from colleagues who have already undertaken this. HIV diagnosis during pregnancy may be a profoundly shocking and life-changing experience for the newly diagnosed

HIV-positive woman. There may be a complex mix of emotional, psychosocial, relationship, economic and even legal issues that Osimertinib arise directly out of the HIV diagnosis. The newly diagnosed woman also has a relatively brief time in which she needs to be able to develop trust in her medical carers and attain sufficient medical knowledge of her situation to be able to make informed decisions that will affect the long-term health of herself, her fetus and her male partner. PMTCT can only be achieved if the pregnant woman embraces medical interventions appropriately. To maximize the effectiveness of interventions for pregnant women in reducing MTCT the psychosocial context of their HIV infection must not be overlooked. filipin Clinical experience indicates that the management of

issues, including dealing with the diagnosis and uncertainty during pregnancy and robust confidentiality processes have an impact on adherence to ART and acceptance of recommended interventions and all clinicians must be mindful of this. 9.1. Antenatal HIV care should be delivered by MDT, the precise composition of which will vary. Grading: 1D The minimum team would comprise an HIV specialist, obstetrician, specialist midwife and paediatrician, with the recommendation of peer- and voluntary-sector support. All efforts should be made to involve the woman’s GP and health visitor. It may be necessary to involve some of the following: patient advocates, social workers, legal advocacy, clinical psychologists, psychiatrists, counsellors, health advisors, Citizens Advice Bureau workers, interpreters, community midwives, clinical nurse specialists and health visitors [313]. In settings with relatively few HIV-positive pregnant women, it is still important to develop robust pathways of care with identified members of an MDT. Regular links, formal or informal, can also be established with a larger unit to provide advice and support as necessary.