Clinical features associated with ADB-BUTINACA exposure in patients attending emergency departments in England.

OBJECTIVE: Synthetic cannabinoid receptor agonists (SCRA) are commonly encountered new psychoactive substances. Here we report the recent detection of ADB-BUTINACA in samples from patients attending United Kingdom emergency departments with toxicity after suspected drug misuse and describe the associated clinical features. METHODS: Consenting adults (≥16 y) presenting to participating hospitals with toxicity after suspected drug misuse have been included in the Identification Of Novel psychoActive substances (IONA) study since March 2015. Demographic and clinical features are recorded and blood and/or urine samples analysed using high-resolution accurate mass liquid chromatography-mass spectrometry. RESULTS: By December 2021, analytical data were available for 1279 IONA participants and ADB-BUTINACA was detected in at least one sample from 10 (9 males, age range 16-51 median 45 years), all presenting since February 2021. Smoking 'spice' was reported by four patients, two had ingested edible "cannabis" gums and four reported heroin use (2 intravenous, 1 smoked, 1 route not known). Co-use of pregabalin (oral) and crack cocaine (smoked) were also reported. In 3 cases ADB-BUTINACA was the only substance detected, while in seven other substances of misuse were also detected including other SCRA, opioids, benzodiazepines cocaine and pregabalin. Clinical features reported in these 2 groups respectively included reduced level of consciousness (3/3, 6/7), agitation (0/3, 4/7), tachycardia (0/3, 3/7), seizures (1/3, 1/7), hallucinations (1/3, 1/7), hypotension (1/3, 1/7). Metabolic acidosis (1/3, 0/7) and respiratory acidosis (1/3, 0/7), All 10 patients recovered with supportive care, including intubation and ventilation for one case. The median length of hospital stay was 19 h (range 2.6-131 h). CONCLUSIONS: ADB-BUTINACA has recently emerged as a drug of misuse in England. Clinical features of toxicity are consistent with those of other SCRA and include reduced level of consciousness, respiratory and/or metabolic acidosis, seizures, confusion and hallucinations.

Methylglyoxal affects cognitive behaviour and modulates RAGE and Presenilin-1 expression in hippocampus of aged mice.

Methylglyoxal (MG), a potent glycotoxin that can be found in the diet, is one of the main precursors of Advanced glycation end products (AGEs). It is well known that modifications in lifestyle such as nutritional interventions can be of great value for preventing brain deterioration. This study aimed to evaluate in vivo how an oral MG treatment, that mimics a high MG dietary intake, could affect brain health. From our results, we demonstrated that MG administration affected working memory, and induced neuroinflammation and oxidative stress by modulating the Receptor for Advanced glycation end products (RAGE). The gene and protein expressions of RAGE were increased in the hippocampus of MG mice, an area where the activity of glyoxalase 1, one of the main enzymes involved in MG detoxification, was found reduced. Furthermore, at hippocampus level, MG mice showed increased expression of proinflammatory cytokines and increased activities of NADPH oxidase and catalase. MG administration also increased the gene and protein expressions of Presenilin-1, a subunit of the gamma-secretase protein complex linked to Alzheimer's disease. These findings suggest that high MG oral intake induces alteration directly in the brain and might establish an environment predisposing to AD-like pathological conditions.

Clinical characteristics, attendance outcomes and deaths of homeless persons in the emergency department: implications for primary health care and community prevention programmes.

OBJECTIVES: Persons experiencing homelessness (PEH) are known to be often excluded from primary health care and community prevention programmes leading to high use of hospital emergency departments (EDs). This study aimed to identify demographic features, clinical characteristics and attendance outcomes of PEH presenting to ED. STUDY DESIGN: Analysis of routinely collected data set. METHODS: Clinical presentations and drug prescription data of PEH who presented a major ED in the West Midlands region of England from 2014 to 2019 were extracted and analysed using descriptive and inferential statistics. RESULTS: During the study period, 3271 of 596,198 presentations were made by PEH; 74% PEH attendees were male. Drug- and alcohol-related conditions, as well as pain and injury constituted the most frequent reasons for presentation, contributing to over half of all presentations. A significantly higher proportion of males (n = 481, 20.3%) presented with drug and alcohol problems than females (n = 93, 11.2%) (P ≤ 0.001). However, pain was the primary reason for presentation for twice as many female patients (n = 189, 22.8%) compared with males (n = 305, 12.9%) (P < 0.001). Nearly one in five left the ED before being assessed and a total of 39 patients (1.2%) died in the ED and 785 (24.0%) required in-patient admissions to the same hospital. CONCLUSIONS: Drug, alcohol and pain including the need of opioid analgesics constituted the majority of presentations made by PEH in ED. The observed rate of death of PEH in ED is 12 times higher than the general population. A very high proportion of PEH also leave the ED before being treated. Future research should focus on strengthening community interventions, particularly to improve access to those at risk of dual diagnoses of substance misuse and mental health problems. Interventions involving multisector collaborations are needed to improve seamless discharge from ED and minimise repeat attendance. Gender differences in the nature of presentations and ED outcomes needs to be investigated further.

A new methodological approach for in vitro determination of the role of DNA methylation on transcription factor binding using AlphaScreen® analysis: Focus on CREB1 binding at hBDNF promoter IV.

BACKGROUND: DNA methylation plays a relevant role in the regulation of gene transcription, but currently the exact quantification of transcription factors binding to methylated DNA is not being determined. The binding of the transcription factor cAMP response element-binding protein-1 to its cognate CpG containing motif is known to be impaired upon methylation. It thus represents a paradigmatic system to experimentally verify the validity of a new in vitro method to measure the role of methylation on DNA/transcription factors binding. METHOD: An AlphaScreen® assay was developed to quantitatively measure the contribution of DNA CpG methylation on the interaction with transcription factors. The method was validated measuring the variation in affinity of cAMP response element-binding protein-1 and its recognition motif in human Brain-derived neurotrophic factor gene exon IV promoter as a function of CpG methylation. RESULTS: For the first time, a quantitative direct correlation between DNA methylation and transcription factors binding is reported showing a dramatic reduction in binding affinity between fully methylated and non-methylated DNA. COMPARISON WITH EXISTING METHODS: This methodology allows to directly measure DNA/transcription factors binding ability as a function of DNA methylation levels thus improving not quantitative methods available today. Moreover, it allows to work with purified proteins and oligonucleotides without need of chromatin. CONCLUSIONS: The present methodology is suggested as a new analytical tool for the quantitative determination of the effect of CpG methylation on the interaction of gene promoters with transcription factors regulating gene expression, a key epigenetic mechanism implicated in many physiological and pathological conditions.

Do intraoperative pyloric interventions predict the need for postoperative endoscopic interventions after minimally invasive esophagectomy?

Intraoperative pyloric procedures are often performed during esophagectomies to reduce the rates of gastric conduit dysfunction. They include pyloroplasty (PP), pyloromyotomy (PM), and pylorus botulinum toxin type-A injections (BI). Despite these procedures, patients frequently warrant further endoscopic interventions. The aim of this study is to compare intraoperative pyloric procedures and the rates of postoperative endoscopic interventions following minimally invasive esophagectomy (MIE). We identified patients who underwent MIE for esophageal carcinoma and grouped them as 'None' (no intervention), 'PP', 'PM', or 'BI' based on intraoperative pyloric procedure type. The rates of endoscopic interventions for the first six postoperative months were compared. To adjust for variability due to MIE type, the rates of >1 interventions were compared using a zero-inflated Poisson regression analysis. Significance was established at P < 0.05. There were 146 patients who underwent an MIE for esophageal cancer from 2008 to 2015; 77.4% were three-hole MIE, and 22.6% were Ivor- Lewis MIE. BI was most frequent in Ivor-Lewis patients (63.5%), while PP was most frequent (46.9%) in three-hole patients. Postoperative endoscopic interventions occurred in 38 patients (26.0%). The BI group had the highest percentage of patients requiring a postoperative intervention (n = 13, 31.7%). After adjusting for higher rates of interventions in three-hole MIE patients, the BI and None groups had the lowest rates of >1 postoperative interventions. Our data did not show superiority of any pyloric intervention in preventing endoscopic interventions. The patients who received BI to the pylorus demonstrated a trend toward a greater likelihood of having a postoperative intervention. However when adjusted for type of MIE, the BI and None groups had lower rates of subsequent multiple interventions. Further research is needed to determine if the choice of intraoperative pyloric procedure type significantly affects quality of life, morbidity, and overall prognosis in these patients.

Detecting non-adherence by urine analysis in patients with uncontrolled hypertension: rates, reasons and reactions.

Poor adherence with pharmacotherapy is well recognised as one of the main barriers to achieving satisfactory blood pressure control, although accurately measuring patient adherence has historically been very challenging. Urine analysis by high-performance liquid chromatography-tandem mass spectrometry has recently become routinely available as a method of screening for non-adherence. In addition to measuring rates of adherence in hypertensive patients, this study aimed to investigate the reasons for non-adherence given by patients and how patients react when they are informed of their results. This was a retrospective observational study looking at results from the routine use of this assay in a specialist hypertension clinic in Birmingham, UK, in patients with uncontrolled hypertension and those under consideration for renal denervation. Out of the 131 patients analysed, only 67 (51%) were taking all their medications as prescribed. Forty-three patients (33%) were taking some of their medications, whilst 21 patients (16%) were completely non-adherent. The most common reasons cited for non-adherence were adverse effects of medication and forgetfulness. Adherence rates for thiazide/thiazide-like diuretics and spironolactone were lower than for other classes of antihypertensive drug. Despite the objective nature and high sensitivity of the test, 36% of non-adherent patients disputed the results. A minority of patients did not attend follow-up. Further research investigating the implications of a 'non-adherence' result on the patient-clinician relationship is required.

Epigenetic modifications of Dexras 1 along the nNOS pathway in an animal model of multiple sclerosis.

The development of multiple sclerosis, a major neurodegenerative disease, is due to both genetic and environmental factors that might trigger aberrant epigenetic changes of the genome. In this study, we analysed global DNA methylation in the brain of mice upon induction of experimental autoimmune encephalomyelitis (EAE), and the effect of environmental enrichment (EE). We demonstrate that global DNA methylation decreased in the striatum, but not in the cortex, of EAE mice compared to healthy controls, in particular in neuronal nitric oxide synthase (nNOS)-positive interneurons of this brain area. Also, in the striatum but again not in the cortex, decreased DNA methylation of the nNOS downstream effector, dexamethasone-induced Ras protein 1 (Dexras 1), was observed in EAE mice, and was paralleled by an increase in its mRNA. Interestingly, EE was able to revert EAE effects on mRNA expression and DNA methylation levels of Dexras 1 and reduced gene expression of nNOS and 5-lipoxygenase (Alox5). Conversely, interleukin-1ß (IL-1ß) gene expression was found up-regulated in EAE mice compared to controls and was not affected by EE. Taken together, these data demonstrate an unprecedented epigenetic modulation of nNOS-signaling in the pathogenesis of multiple sclerosis, and show that EE can specifically revert EAE effects on Dexras 1 along this pathway.

A low-cost and versatile system for projecting wide-field visual stimuli within fMRI scanners.

We have constructed and tested a custom-made magnetic-imaging-compatible visual projection system designed to project on a very wide visual field (~80°). A standard projector was modified with a coupling lens, projecting images into the termination of an image fiber. The other termination of the fiber was placed in the 3-T scanner room with a projection lens, which projected the images relayed by the fiber onto a screen over the head coil, viewed by a participant wearing magnifying goggles. To validate the system, wide-field stimuli were presented in order to identify retinotopic visual areas. The results showed that this low-cost and versatile optical system may be a valuable tool to map visual areas in the brain that process peripheral receptive fields.

Tiempos posibles de la transferencia en una adolescente: efectos en la subjetividad / Possible times of an adolescent transference: effects on subjectivity

El presente desarrollo se inscribe en la investigación "Modalidades de las transferencias de pacientes adolescentes que han sido abusados sexualmente en la infancia" 2013-2016.A partir de un caso de la práctica clínica se intenta dar cuenta de la apertura y devenir de la transferencia que, sostenida en el lazo analizante-analista, devela la continuidad-discontinuidad de los tiempos necesarios y propicios para que poco a poco lo disociado-silenciado que conlleva lo traumático: el abuso sexual vivido alrededor de los 8 años de edad, tenga lugar. Eso traumático se presenta como reviviscencia, no como recuerdo. Lo escindido (no reprimido) retorna y algo nuevo se produce: el abrazo y la mirada de la analista marca -como intervención en transferencia, el procesamiento de eso que era y sigue siendo tan excesivo.

This development is a part of the investigation "Transference Modalities of adolescent patients who have been sexually abused in childhood". From a clinical practice case, it is attemped to account of the opening and future of the transference. This phenomenon, when sustainedin the analysand-analyst link, reveals the continuity-discontinuity of the necessary and adequate times for the disassociated-silenced connected to the traumatic to take place little by little; that is, the sexual abuse experience around the age of eight. That traumatic episode is presented as a lashback, not as a memory. What is divided (not repressed) returns and something new takes place: the hug and look of the analyst marks - as intervention in transference- the processing of what was and remains so excessive.

Endocannabinoid signaling and food addiction.

Overeating, frequently linked to an increasing incidence of overweight and obesity, has become epidemic and one of the leading global health problems. To explain the development of this eating behavior, new hypotheses involve the concept that many people might be addicted to food by losing control over their ability to regulate food intake. Among the different neurotransmitter networks that partake in the reward circuitry within the brain, a large body of evidence supports the involvement of the endocannabinoid system. Indeed, its dysfunctions might contribute to food addiction, by regulating appetite and food preference through central and peripheral mechanisms. Here, we review and discuss the role of endocannabinoid signaling in the reward circuitry, and the possible therapeutic exploitation of strategies based on its fine regulation.

Abuso sexual infantil y el investimento: desinvestimento libidinal del cuerpo sexuado genitalmente / Child abuse and the libidinization: delibidinization of the genitally sexed body

La presente investigación se propone evaluar cuáles son los efectos del abuso sexual infantil en la apropiación subjetiva de la sexualidad genital de adolescentes que han sido abusados sexualmente en la infancia. Para ello, nos proponemos: explorar la relación entre el abuso sexual infantil y el investimento/desinvestimento libidinal del cuerpo sexuado genitalmente. En el marco de un diseño exploratorio, se ha trabajado con una muestra finalística conformada por siete adolescentes que han sido abusados sexualmente en la infancia y que realizan o han realizado tratamiento psicoterapéutico. La fuente de datos han sido los materiales clínicos elaborados por los psicoanalistas a cargo del tratamiento de cada adolescente. El procedimiento de análisis ha sido cualitativo, de tipo hermenéutico. En el análisis de estos materiales clínicos nos abocamos a analizar los trabajos psíquicos que realizaron los adolescentes para investir libidinalmente el cuerpo sexuado genitalmente, y las dificultades para que se desplegara dicho proceso.(AU)

This research has the goal of assessing the effects of child abuse in the subjective appropriation of genital sexuality in adolescents who have been sexually abused in their childhood. To accomplish this, we will: explore the link between child abuse and the libidinization/delibidinization of the genitally sexed body.Using an exploratory framework, we have worked with a sample of seven adolescents who have been sexually abused in their childhood and have undergone psychotherapeutic treatment. The source for our data were the clinical records kept by the psychoanalysts who lead each adolescentÆs treatment.In the assessment of these clinical records we focused in the analysis of the psychic works done by the adolescents in order to libidinize the genitally sexed body, and the difficulties in deploying such process. (AU)

Abuso sexual infantil y el investimento: desinvestimento libidinal del cuerpo sexuado genitalmente / Child abuse and the libidinization: delibidinization of the genitally sexed body

La presente investigación se propone evaluar cuáles son los efectos del abuso sexual infantil en la apropiación subjetiva de la sexualidad genital de adolescentes que han sido abusados sexualmente en la infancia. Para ello, nos proponemos: explorar la relación entre el abuso sexual infantil y el investimento/desinvestimento libidinal del cuerpo sexuado genitalmente. En el marco de un diseño exploratorio, se ha trabajado con una muestra finalística conformada por siete adolescentes que han sido abusados sexualmente en la infancia y que realizan o han realizado tratamiento psicoterapéutico. La fuente de datos han sido los materiales clínicos elaborados por los psicoanalistas a cargo del tratamiento de cada adolescente. El procedimiento de análisis ha sido cualitativo, de tipo hermenéutico. En el análisis de estos materiales clínicos nos abocamos a analizar los trabajos psíquicos que realizaron los adolescentes para investir libidinalmente el cuerpo sexuado genitalmente, y las dificultades para que se desplegara dicho proceso.

This research has the goal of assessing the effects of child abuse in the subjective appropriation of genital sexuality in adolescents who have been sexually abused in their childhood. To accomplish this, we will: explore the link between child abuse and the libidinization/delibidinization of the genitally sexed body.Using an exploratory framework, we have worked with a sample of seven adolescents who have been sexually abused in their childhood and have undergone psychotherapeutic treatment. The source for our data were the clinical records kept by the psychoanalysts who lead each adolescent’s treatment.In the assessment of these clinical records we focused in the analysis of the psychic works done by the adolescents in order to libidinize the genitally sexed body, and the difficulties in deploying such process.

Growth factors, their receptor expression and markers for proliferation of endothelial and neoplastic cells in human osteosarcoma.

Osteosarcoma is the most common primary malignant tumour of the bone. Although new therapies continue to be reported, osteosarcoma-related morbidity and mortality remain high. Modern medicine has greatly increased knowledge of the physiopathology of this neoplasm. Novel targets for drug development may be identified through an understanding of the normal molecular processes that are deeply modified in pathological conditions. The aim of the present study is to investigate, by immunohistochemistry, the localisation of different growth factors and of the proliferative marker Ki-67 in order to determine whether these factors are involved in the transformation of osteogenic cells and in the development of human osteosarcoma. We observed a general positivity for NGF - TrKA - NT3 - TrKC - VEGF in the cytoplasm of neoplastic cells and a strong expression for NT4 in the nuclear compartment. TGF-beta was strongly expressed in the extracellular matrix and vascular endothelium. BDNF and TrKB showed a strong immunolabeling in the extracellular matrix. Ki-67/MIB-1 was moderately expressed in the nucleus of neoplastic cells. We believe that these growth factors may be considered potential therapeutic targets in the treatment of osteosarcoma, although proof of this hypothesis requires further investigation.

Endothelial cells and normal breast epithelial cells enhance invasion of breast carcinoma cells by CXCR-4-dependent up-regulation of urokinase-type plasminogen activator receptor (uPAR, CD87) expression.

Here we show the increase of invasion of three breast cancer cell lines (8701-BC, MDA-MB-231 and SKBR3) upon long-term co-incubation with culture medium of normal microvascular endothelial cells (MVEC) and normal breast epithelial cells (HB2). The enhancement of invasion relied on the interaction of microvascular endothelial cell and normal breast epithelial cell CXCL12 (SDF1) chemokine, whose expression by breast cancer cells was very low, with the cognate CXCR4 receptor of malignant cells, which resulted in over-expression of the urokinase-type plasminogen activator receptor (uPAR) on their surfaces. uPAR over-expression, showed by RT-PCR and Western blotting, was paralleled by increased urokinase-type plasminogen activator (uPA) partitioning on the cell surface with respect to the fluid phase, as demonstrated by zymography. Long-term interaction of SDF1 with CXCR4 stimulated sustained activation of JNK phosphorylation. Blocking antibodies to CXCR4 were able to block the endothelial/epithelial cell-dependent enhancement of invasion, as well as to inhibit SDF1-CXCR4-dependent JNK phosphorylation and uPAR over-expression of malignant cells. We suggest that acquisition of the angiogenic phenotype by breast cancer cells triggers an amplification loop, in which endothelial cells and normal breast epithelial cells of the tumour cooperate to provide facilitated routes to cell invasion and metastasis and to enhance the aggressive phenotype of cancer cells.

Remediation of waters contaminated with ionic herbicides by sorption on polymerin.

This study investigated the sorption of paraquat and 2,4-D on polymerin, the humic acid-like fraction of olive mill wastewater. Effects of pH, contact time, initial concentration and sorbent dosage on the sorption of both herbicides were studied. The sorption mechanism of paraquat on polymerin was consistent with the ion exchange of this herbicide with Ca, Mg and K natively occurring in the sorbent; in contrast, 2,4-D was bound to polymerin by hydrogen bonding. Simulated wastewaters contaminated with paraquat were purified after three sorption cycles on polymerin renewed at each cycle, at a solid/liquid ratio of 0.5, whereas those containing 2,4-D showed a maximal residue removal of 44% after two sorption cycles at the same ratio. The possible application of this model to other water-soluble herbicides, as well as the possible exploitation of polymerin as a bio-filter for the decontamination of pollution point sources is briefly discussed.

Systemic sclerosis fibroblasts inhibit in vitro angiogenesis by MMP-12-dependent cleavage of the endothelial cell urokinase receptor.

Failure of endothelial cells to develop new vessels in response to hypoxia is a distinctive feature of systemic sclerosis (SSc) in the avascular phase. We have previously shown that SSc endothelial cells over-express matrix metalloproteinase-12 (MMP-12), which blocks angiogenesis by cleavage of the endothelial urokinase-type plasminogen activator receptor (uPAR). In the present study, we have investigated whether over-expression of MMP-12 and of angiostatic factors, or hypo-expression of angiogenic factors by SSc fibroblasts, contributes to impaired angiogenesis in SSc. Dermal fibroblasts were isolated from healthy subjects (N-Fb) and patients with diffuse SSc (SSc-Fb). Angiogenesis of target normal human microvascular endothelial cells (H-MVECs) was assayed by Matrigel invasion, cell proliferation, and capillary morphogenesis. uPAR cleavage and MMP-12 activity were evaluated by western blotting. We show that the over-expression of MMP-12 by SSc-Fb determines uPAR cleavage in H-MVECs. Conditioned medium from SSc-Fb impaired H-MVEC proliferation, invasion, and capillary morphogenesis. Anti-MMP-12 antibodies restored such impairment. Altered expression of angiostatic/angiogenic factors, including transforming growth factor beta1, did not account for SSc-Fb-dependent impairment of angiogenesis. The over-expression of MMP-12 by both SSc-Fb and SSc endothelial cells indicates that MMP-12 over-production may have a critical pathogenic role in SSc-associated vascular alterations.

Effects of blocking urokinase receptor signaling by antisense oligonucleotides in a mouse model of experimental prostate cancer bone metastases.

An important factor implicated in tumor cell predisposition for invasion and metastasis is the malignancy-related upregulation of urokinase plasminogen activator receptor (uPAR). uPAR signals by activating different tyrosine kinases in different cells. We examined the effects of inhibiting uPAR signaling by inhibition of uPAR expression with antisense oligonucleotides (aODNs) in PC3 human prostate cancer cells and evaluated aODN effect in a mouse model of prostate cancer bone metastasis. Following uPAR aODN treatment, PC3 cells exhibited a strong decrease in uPAR expression, evaluated by flow cytometry and by polymerase chain reaction, and of FAK/JNK/Jun phosphorylation. The synthesis of cyclins A, B, D1 and D3 was inhibited, as shown by Western blotting, flow cytometry and polymerase chain reaction, and PC3 cells accumulated in the G2 phase of the cell cycle. PC3 cells' adhesion was unaffected, while proliferation and invasion of Matrigel were impaired. A total of 60 mice were subjected to intracardiac injection of PC3 cells and were randomly assigned to three groups: aODN (treated with 0.5 mg intraperitoneum/mouse/day), dODN (treated with the same amounts of a degenerated ODN) and control (injected with a saline solution). At 28 days after heart injection, mice were subjected to a digital scan of total body radiography, which revealed 80% reduction in mice affected by bone metastasis. The use of uPAR aODNs produced a substantial prophylactic effect against prostate cancer bone metastasis, which has to be ascribed to downregulation of uPAR expression.

Role of penicillin-binding protein 4 in expression of vancomycin resistance among clinical isolates of oxacillin-resistant Staphylococcus aureus.

It has been reported that penicillin-binding protein 4 (PBP4) activity decreases when a vancomycin-susceptible Staphylococcus aureus isolate is passaged in vitro to vancomycin resistance. We analyzed the PBP profiles of four vancomycin intermediately susceptible S. aureus (VISA) clinical isolates and found that PBP4 was undetectable in three isolates (HIP 5827, HIP 5836, and HIP 6297) and markedly reduced in a fourth (Mu50). PBP4 was readily visible in five vancomycin-susceptible, oxacillin-resistant S. aureus (ORSA) isolates. The nucleotide sequences of the pbp4 structural gene and flanking sequences did not different between the VISA and vancomycin-susceptible isolates. Overproduction of PBP4 on a high-copy-number plasmid in the VISA isolates produced a two- to threefold decrease in vancomycin MICs. Inactivation of pbp4 by allelic replacement mutagenesis in three vancomycin-susceptible ORSA strains (COL, RN450M, and N315) led to a decrease in vancomycin susceptibility, an increase in highly vancomycin-resistant subpopulations, and decreased cell wall cross-linking by high-performance liquid chromatography analysis. Complementation of the COL mutant with plasmid-encoded pbp4 restored the vancomycin MIC and increased cell wall cross-linking. These data suggest that alterations in PBP4 expression are at least partially responsible for the VISA phenotype.