Multi-focal Stimulation of the Cortico-cerebellar Loop During the Acquisition of a Novel Hand Motor Skill in Chronic Stroke Survivors.

Impairment of hand motor function is a frequent consequence after a stroke and strongly determines the ability to regain a self-determined life. An influential research strategy for improving motor deficits is the combined application of behavioral training and non-invasive brain stimulation of the motor cortex (M1). However, a convincing clinical translation of the present stimulation strategies has not been achieved yet. One alternative and innovative approach is to target the functionally relevant brain network-based architecture, e.g., the dynamic interactions within the cortico-cerebellar system during learning. Here, we tested a sequential multifocal stimulation strategy targeting the cortico-cerebellar loop. Anodal transcranial direct current stimulation (tDCS) was applied simultaneously to a hand-based motor training in N = 11 chronic stroke survivors during four training sessions on two consecutive days. The tested conditions were: sequential multifocal (M1-cerebellum (CB)-M1-CB) vs. monofocal control stimulation (M1-sham-M1-sham). Additionally, skill retention was assessed 1 and 10 days after the training phase. Paired-pulse transcranial magnetic stimulation data were recorded to characterize stimulation response determining features. The application of CB-tDCS boosted motor behavior in the early training phase in comparison to the control condition. No faciliatory effects on the late training phase or skill retention were detected. Stimulation response variability was related to the magnitude of baseline motor ability and short intracortical inhibition (SICI). The present findings suggest a learning phase-specific role of the cerebellar cortex during the acquisition of a motor skill in stroke and that personalized stimulation strategies encompassing several nodes of the underlying brain network should be considered.

Thickness uniformity measurements and damage threshold tests of large-area GaAs/AlGaAs crystalline coatings for precision interferometry.

Precision interferometry is the leading method for extremely sensitive measurements in gravitational wave astronomy. Thermal noise of dielectric coatings poses a limitation to the sensitivity of these interferometers. To decrease coating thermal noise, new crystalline GaAs/AlGaAs multilayer mirrors have been developed. To date, the surface figure and thickness uniformity of these alternative low-loss coatings has not been investigated. Surface figure errors, for example, cause small angle scattering and thereby limit the sensitivity of an interferometer. Here we measure the surface figure of highly reflective, substrate-transferred, crystalline GaAs/AlGaAs coatings with a custom scanning reflectance system. We exploit the fact that the reflectivity varies with the thickness of the coating. To increase penetration into the coating, we used a 1550 nm laser on a highly reflective coating designed for a center wavelength of 1064 nm. The RMS thickness variation of a two inch optic was measured to be 0.41 ± 0.05 nm. This result is within 10% of the thickness uniformity, of 0.37 nm RMS, achieved with ion-beam sputtered coatings for the aLIGO detector. We additionally measured a lower limit of the laser induced damage threshold of 64 MW/cm 2 for GaAs/AlGaAs coatings at a wavelength of 1064 nm.

Generation of functional human serotonergic neurons from fibroblasts.

The brain's serotonergic system centrally regulates several physiological processes and its dysfunction has been implicated in the pathophysiology of several neuropsychiatric disorders. While in the past our understanding of serotonergic neurotransmission has come mainly from mouse models, the development of pluripotent stem cell and induced fibroblast-to-neuron (iN) transdifferentiation technologies has revolutionized our ability to generate human neurons in vitro. Utilizing these techniques and a novel lentiviral reporter for serotonergic neurons, we identified and overexpressed key transcription factors to successfully generate human serotonergic neurons. We found that overexpressing the transcription factors NKX2.2, FEV, GATA2 and LMX1B in combination with ASCL1 and NGN2 directly and efficiently generated serotonergic neurons from human fibroblasts. Induced serotonergic neurons (iSNs) showed increased expression of specific serotonergic genes that are known to be expressed in raphe nuclei. iSNs displayed spontaneous action potentials, released serotonin in vitro and functionally responded to selective serotonin reuptake inhibitors (SSRIs). Here, we demonstrate the efficient generation of functional human serotonergic neurons from human fibroblasts as a novel tool for studying human serotonergic neurotransmission in health and disease.

Occupational skin diseases: actual state analysis of patient management pathways in 28 European countries.

BACKGROUND: Work-related skin diseases (WSD) are caused or worsened by a professional activity. Occupational skin diseases (OSD) need to fulfil additional legal criteria which differ from country to country. OSD range amongst the five most frequently notified occupational diseases (musculoskeletal diseases, neurologic diseases, lung diseases, diseases of the sensory organs, skin diseases) in Europe. OBJECTIVE: To retrieve information and compare the current state of national frameworks and pathways to manage patients with occupational skin disease with regard to prevention, diagnosis, treatment and rehabilitation in different European countries. METHODS: A questionnaire-based survey of the current situation regarding OSD patient management pathways was carried out with experts on occupational dermatology and/or occupational medicine from 28 European countries contributing to the European Cooperation in Science and Technology (COST) Action TD 1206 (StanDerm) (www.standerm.eu). RESULTS: Besides a national health service or a statutory health insurance, most European member states implemented a second insurance scheme specifically geared at occupational diseases [insurance against occupational risks (synonyms: insurance against work accidents and occupational injuries; statutory social accident insurance)]. Legal standards for the assessment of occupationally triggered diseases with a genetic background differ between different countries, however, in most European member states recognition as OSD is possible. In one-third of the countries UV light-induced tumours can be recognized as OSD under specific conditions. CONCLUSION: OSD definitions vary between European countries and are not directly comparable, which hampers comparisons between statistics collected in different countries. Awareness of this fact and further efforts for standardization are necessary.

[Single center analysis of the dermatosurgical patient cohort of a tumor center in Germany]. / Betrachtung des dermatochirurgischen Patientenkollektivs an einem Hauttumorzentrum in Deutschland.

BACKGROUND: The incidence of skin cancer continues to increase. However, little is known about the dermatosurgical characteristics of the patients. PATIENTS AND METHODS: In this single center, retrospective study, dermatosurgical reports of all patients treated because of basal cell carcinomas (BCC), squamous cell carcinomas (SCC), and malignant melanoma (MM) between 2004 and 2013 were analyzed. RESULTS: During the observed period, the number of operated BCC rose by a factor of 1.86 and the number of MM by a factor of 2.3. In comparison to BCC/MM, there was a disproportionately high increase of SCC by a factor of 4.02. The average age was 71.5 ± 13.4 years (minimum: 14 years; maximum: 104 years), whereupon a significant increase of male age and a significant decrease of female age occurred. Almost 70% of all tumors were located in the head and neck area. The nose was most commonly treated. CONCLUSIONS: During the last 10 years, the cohort of dermatosurgical patients changed in the tumor center. This should be verified in multicenter studies.

[Imaging Blood Flow and Pulsation of Retinal Vessels with Full-Field Swept-Source OCT]. / Darstellung von Blutfluss und Pulsation in retinalen Gefäßen mit Full-Field-Swept-Source-OCT.

Optical coherence tomography (OCT) uses interference to image the retina with high axial resolution. In the last 25 years, new technologies have permitted a steady increase in imaging speed, which made it possible to enlarge the imaged field and to avoid motion artefacts. The speed and precision of retinal imaging is now limited by photodamage of the retina caused by the focused OCT beam and by the speed of the scanning mechanics. Full-field swept-source (FF-SS)-OCT decreases irradiance on the retina and dispenses moving parts by using a camera to acquire the full volume of the retina in parallel. Here we show that FF-SS-OCT is rapid and precise enough to image pulsation in the retina induced by the heart beat. Series of OCT volumes 1.8 × 0.7 mm wide and 1.8 mm deep were recorded in young volunteers over a few cycles of the heart beat. Morphology of the retinal vessels, blood flow and tissue motion as caused by vessel pulsation were calculated from the OCT data. FF-SS-OCT was able to visualise the main structures of the neuronal retina, including vessels and small capillaries and without any motion artefacts. Information on three different dynamic processes was obtained from only one recorded series of OCT volumes: pulsation of blood flow and blood pressure in retinal vessels as well as pulsation of the choroid. Delays between arterial and venous pulse and delay between pulsation in retinal and choroidal vessels were calculated. With a time resolution of 0.5 ms, FF-SS-OCT is able to visualise previously unmeasurably fast changes in the retina, including the propagation of pulse waves.

Radiographic accuracy in TKA with a CT-based patient-specific cutting block technique.

PURPOSE: Patient-specific instrumentation (PSI) technology for the implantation of total knee arthroplasty (TKA) has a rising interest in the orthopaedic community. Data of PSI are controversially discussed. The hypothesis of this paper is that the radiological accuracy of CT-based PSI is similar to the one of navigated TKA published in the literature. METHODS: Since 2010, all 301 consecutively performed PSI TKAs (GMK MyKnee©) were included in this study. The radiological assessment consisted in a preoperative and postoperative standard X-ray and long-standing X-ray. Changes from the planned to the definitively implanted component size were documented. Postoperative analysis included limb alignment and position of femoral and tibial components (for varus/valgus and flexion or tibial slope). RESULTS: The postoperative average hip-knee-ankle angle was 180.1° ± 2.0°. In the frontal plane a total of 12.4 % of outliers >3°, for the tibial components 4.1 % of outliers >3° and for the femoral components 4.8 % of outliers >3° were measured. A total of 12.3 % of outliers for posterior tibial slope and 9 % of outliers >3° for the femoral flexion were noted. 10.8 % of the 602 planned size components were adapted intraoperatively. CONCLUSION: Although it is still unknown which limb axis is the correct one for the best clinical result, a technology providing the aimed axis in a most precise way should be chosen. Comparing the outcome of the current study with the data from the literature, there does not seem to be any difference compared to computer-assisted surgery. LEVEL OF EVIDENCE: IV.

Bilateral diffuse pigmented villonodular synovitis of the knee.

Pigmented villonodular synovitis (PVNS) is a proliferative disease of synovial tissue characterized by lipid-laden macrophages, multinucleated giant cells, and hemosiderin deposits. PVNS presents either in a localized form with minimal rates of recurrence after surgical resection or in a diffuse form with an expansive growth pattern showing formation of osseous erosions and extra-articular manifestation. In the diffuse form high recurrence rates occur as a result of the challenge of achievement of total synovectomy. Typically only one single joint, being the knee in 80% of cases, is involved with diffuse PVNS. Reports of bi- or multiarticular manifestation are at best rare. Here, a case of a 16-year-old girl with bilateral diffuse PVNS of the knee allows discussion of diagnostic and treatment considerations.

Vitrectomy in uveitis patients.

PURPOSE: To evaluate the causes and success rates of pars plana vitrectomy (PPV) in uveitis patients. METHODS: Retrospective study of the charts of 26 uveitis patients (28 eyes) who underwent PPV between the years 2008 and 2011. We examined surgical indications and success rates, based on visual outcomes, complications and diagnosis in case of vitreous biopsy. RESULTS: (1) Therapeutic PPV (TV) was performed in 36% of the eyes, (2) TV combined with epiretinal membrane (ERM) peeling in 21% and (3) diagnostic PPV (DiV) was performed in 64% of the eyes. Eight eyes (28,6%) underwent a combined cataract and vitreous surgery. Visual acuity (VA) improved in 16 eyes (57%), with a mean improvement of -0,9 log of the minimum angle of resolution (logMAR), although the effect was transient in 7% of the cases. VA remained stable in 11 eyes (39%) and decreased in 1 (4%). Post-operative complications were cystic macular oedema (CMO) in 3 eyes (11%), cataract in 5 eyes (18%) and retinal detachment in 2 eyes (7%). Diagnostic tests were performed in 18 eyes with a success rate of 55%. CONCLUSIONS: In our series of patients with uveitis, a good andstable improvement of VA was found when PPV was performed with ERM peeling while the effect on VA was more transient in the other cases. A good success rate of diagnosis was also found in DiV. However, considering the possible severe complications, diagnostic vitrectomy should be limited to selected cases.

Stable isotope analyses reveal individual variability in the trophic ecology of a top marine predator, the southern elephant seal.

Identifying individuals' foraging strategies is critical to understanding the ecology of a species, and can provide the means to predict possible ecological responses to environmental change. Our study combines stable isotope analysis and satellite telemetry to study the variability in individual foraging strategies of adult female southern elephant seals (Mirounga leonina). Our hypothesis is that female elephant seals from the Western Antarctica Peninsula (WAP) display individual specialization in their diets. We captured adult female elephant seals (n = 56, 2005-2009) at Livingston Island (Antarctica), and instrumented them with SMRU-CTD satellite tags. We collected blood, fur, and vibrissae samples for δ(13)C and δ(15)N analyses. The mean values for all vibrissae were -21.0 ± 0.7‰ for δ(13)C, and 10.4 ± 0.8‰, for δ(15)N. The individual variability of δ(13)C (60%) was more important than the within-individual variability (40%) in explaining the total variance observed in our data. For δ(15)N, the results showed the opposite trend, with the within-individual variability (64%) contributing more to the total variance than the individual variability (36%), likely associated with the effect that the fasting periods have on δ(15)N values. Most individuals were specialists, as inferred from the low intra-individual variability of δ(13)C values with respect to the population variability, with half the individuals utilizing 31% or less of their available niche. We found eight different foraging strategies for these animals. Female elephant seals from the WAP are a diverse group of predators with individuals utilizing only a small portion of the total available niche, with the consequent potential to expand their foraging habits to exploit other resources or environments in the Southern Ocean.

Desmosomal cadherins: another growing multigene family of adhesion molecules.

The formation of supracellular structures, i.e. tissues and organs, is dependent on the spatially and temporally regulated formation of semistable cell-cell contacts. In recent years, the molecular components of such cell junctions, especially those occurring in epithelial cells, have been studied extensively, and the main proteins and glycoproteins of the 'adhering junctions' such as the desmosomes and the zonula adherens of polar epithelial cells have been characterized. We are now beginning to understand the complex protein-protein interactions that contribute to the assembly and disassembly of these structures and their roles in the attachment of specific filaments of the cytoskeleton.

Is noncontact ACL injury associated with the posterior tibial and meniscal slope?

BACKGROUND: The risk of noncontact ACL injury reportedly is increased in patients with a greater posterior tibial slope (PTS), but clinical data are inconsistent. It is unclear whether the medial and lateral PTSs have a different impact on this connection. It also is unknown whether the meniscal slope (MS) is associated with ACL injury. PATIENTS/METHODS: Using MRI, we compared the medial and lateral PTSs and MSs separately in 55 matched pairs of patients with isolated noncontact ACL injuries and a control group. RESULTS: Neither the PTS nor the relative difference between the medial and lateral PTSs differed between groups. In contrast, the lateral MS was greater with ACL injuries: 2.0° versus -2.7° in males with and without ACL injury and 1.7° versus -0.9 in females. Uninjured females had a greater PTS than males: 4.9° versus 3.0° in females and males medially, respectively; 5.7° versus 4.0° lateral. CONCLUSIONS: There is no obvious link between the medial or lateral PTSs and ACL injury, and there is no obvious link between the relative difference in the medial and lateral PTSs and noncontact ACL injury. However, a greater lateral MS may indicate a greater risk of injury. The PTS can differ between the genders but the average difference is small. LEVEL OF EVIDENCE: Level II, prognostic study. See the Guidelines for Authors for a complete description of levels of evidence.

Bubble dynamics in a standing sound field: the bubble habitat.

Bubble dynamics is investigated numerically with special emphasis on the static pressure and the positional stability of the bubble in a standing sound field. The bubble habitat, made up of not dissolving, positionally and spherically stable bubbles, is calculated in the parameter space of the bubble radius at rest and sound pressure amplitude for different sound field frequencies, static pressures, and gas concentrations of the liquid. The bubble habitat grows with static pressure and shrinks with sound field frequency. The range of diffusionally stable bubble oscillations, found at positive slopes of the habitat-diffusion border, can be increased substantially with static pressure.

Evaluation of Novel Substituted Furopyridines as Inhibitors of Protein Kinases Related to Tau Pathology in Alzheimer´s Disease.

BACKGROUND: Alzheimer´s disease (AD) is characterized by a progressive neuronal degeneration caused by two pathological hallmarks, hyperphosphorylated tau protein aggregated into tau filaments and amyloid precursor protein derived beta amyloid peptides aggregated into extracellular amyloid plaques. All attempts so far to find effective drugs failed in clinical trials. AD is a multifactorial disease, so that selective drugs to target one AD-relevant structure alone may not be sufficient. OBJECTIVE: We built novel furopyridines with various substitution patterns to evaluate them as protein kinases inhibitors of enzymes related to tau pathology. METHODS: Furopyridine derivatives were synthesized and purified using column chromatography. The protein kinase inhibitory properties were determined in ATP-competition assays with determined affinity constants for the most active compounds. RESULTS: The compounds were prepared in simple two-component reactions of substituted 1,4- dihydropyridines and respective quinones to obtain various substitutions of the molecular furopyridine scaffold. The substituent effects on the determined kinase inhibitory properties of cdk1, cdk2, Fyn, JNK3 and gsk-3ß are discussed. CONCLUSION: Various 3-substitutions were found most sensitive for the protein kinase inhibition depending on the length, nature and a substituent positioning within. We identified compounds as inhibitors of several kinases as a tool to potentially combat the disease progress in a multitargeting approach.

Lytic bone lesions in lupus-associated myelofibrosis.

Myelofibrosis is associated with numerous causes other than hematologic malignant neoplasms, including autoimmune diseases. We describe a 44-year-old woman who suffered bone lytic lesions with extramedullary haematopoiesis in the setting of myelofibrosis associated with systemic lupus erythematosus.

First Report of Brown Ring Patch Caused by Waitea circinata var. circinata on Poa annua in Wisconsin and Minnesota.

In summer of 2008, two turfgrass samples were submitted to the Turfgrass Diagnostic Lab at the University of Wisconsin-Madison. The samples were from golf courses in Beaver Dam, WI on 12 June and Minneapolis, MN on 14 July. Both samples were collected from 40-year-old native soil putting greens mowed at 3.2 mm that had received annual sand topdressing since 1992. The putting greens were a mixture of approximately 75% annual bluegrass (Poa annua L.) and 25% creeping bentgrass (Agrostis stolonifera L.) Stand symptoms observed in the field were bright yellow, sunken rings that were approximately 5 cm thick and 15 to 35 cm in diameter. Some rings were incomplete, giving a scalloped appearance. Affected plants were severely chlorotic and lacked any discrete lesions or spots. Symptoms were more prominent on annual bluegrass than creeping bentgrass. Upon incubation of samples at room temperature in a moist chamber for 24 h, fungal mycelia with septations and right-angle branching were observed in the foliage and thatch layer. Two isolates were obtained from affected annual bluegrass in each sample. Isolations were performed by washing affected leaves in 0.5% NaOCl solution for 2 min, blotting the tissue dry, and plating the tissue on potato dextrose agar (PDA) amended with chloramphenicol (0.05 g/liter), streptomycin (0.05 g/liter), and tetracycline (0.05 g/liter). After incubation for 2 days at 23°C, isolates were transferred and maintained on PDA. All four isolates had multinucleate hyphae and displayed sclerotial characteristics similar to those reported for Waitea circinata var. circinata (2). Sequencing the ITS1F/ITS4-amplified rDNA internal transcribed spacer (ITS) region confirmed the isolates as W. circinata var. circinata, with ≥99% sequence similarity to published W. circinata var. circinata ITS sequences (GenBank Accession No. FJ755849) (1,2,4). To confirm pathogenicity, isolates were inoculated onto 6-week-old annual bluegrass (True Putt/DW184) grown in 10-cm-diameter pots containing calcined clay (Turface; Profile Products LLC., Buffalo Grove, IL). Two 4-mm-diameter agar plugs for each isolate were removed from the margins of 3-day-old colonies grown on PDA and placed near the soil surface to ensure contact with the lower leaf blades. Each isolate was placed in four separate pots to have four replicated tests per isolate, and four noninfested pots were utilized as negative controls. All pots were placed in moist chambers at 28°C with a 12-h light/dark cycle. Within 4 to 6 days, inoculated plants exhibited severe chlorosis and a minor amount of aerial mycelium was observed. Inoculated plants became necrotic after 15 to 20 days, while the noninoculated plants remained healthy. W. circinata var. circinata was reisolated from inoculated plants and its identity was confirmed by morphological and molecular characteristics. This pathogen was previously reported as a causal agent of brown ring patch of creeping bentgrass in Japan and annual bluegrass in the western United States (2,4). To our knowledge, this is the first report of brown ring patch in Minnesota and Wisconsin. Intensive fungicide practices are needed to control brown ring patch; therefore, this disease could have significant economic impact throughout the Upper Midwest (3). References: (1) C. M. Chen et al. Plant Dis. 93:906, 2009 (2) K. de la Cerda et al. Plant Dis. 91:791, 2007. (3) J. Kaminski and F. Wong. Golf Course Manage. 75(9):98, 2007. (4) T. Toda et al. Plant Dis. 89:536, 2005.

The localization of bullous pemphigoid antigen 180 (BP180) in hemidesmosomes is mediated by its cytoplasmic domain and seems to be regulated by the beta4 integrin subunit.

Bullous pemphigoid antigen 180 (BP180) is a component of hemidesmosomes, i.e., cell-substrate adhesion complexes. To determine the function of specific sequences of BP180 to its incorporation in hemidesmosomes, we have transfected 804G cells with cDNA-constructs encoding wild-type and deletion mutant forms of human BP180. The results show that the cytoplasmic domain of BP180 contains sufficient information for the recruitment of the protein into hemidesmosomes because removal of the extracellular and transmembrane domains does not abolish targeting. Expression of chimeric proteins, which consist of the membrane targeting sequence of K-Ras fused to the cytoplasmic domain of BP180 with increasing internal deletions or lacking the NH2 terminus, indicates that the localization of BP180 in hemidesmosomes is mediated by a segment that spans 265 amino acids. This segment comprises two important regions located within the central part and at the NH2 terminus of the cytoplasmic domain of BP180. To investigate the effect of the alpha6beta4 integrin on the subcellular distribution of BP180, we have transfected COS-7 cells, which lack alpha6beta4 and BP180, with cDNAs for BP180 as well as for human alpha6A and beta4. We provide evidence that a mutant form of BP180 lacking the collagenous extracellular domain as well as a chimeric protein, which contains the entire cytoplasmic domain of BP180, are colocalized with alpha6beta4. In contrast, when cells were transfected with cDNAs for alpha6A and mutant forms of beta4, either lacking the cytoplasmic COOH-terminal half or carrying phenylalanine substitutions in the tyrosine activation motif of the cytoplasmic domain, the recombinant BP180 molecules were mostly not colocalized with alpha6beta4, but remained diffusely distributed at the cell surface. Moreover, in cells transfected with cDNAs for alpha6A and a beta4/beta1 chimera, in which the cytoplasmic domain of beta4 was replaced by that of the beta1 integrin subunit, BP180 was not colocalized with the alpha6beta4/beta1 chimera in focal adhesions, but remained again diffusely distributed. These results indicate that sequences within the cytoplasmic domain of beta4 determine the subcellular distribution of BP180.

Transgenic mice expressing a mutant form of loricrin reveal the molecular basis of the skin diseases, Vohwinkel syndrome and progressive symmetric erythrokeratoderma.

Mutations in the cornified cell envelope protein loricrin have been reported recently in some patients with Vohwinkel syndrome (VS) and progressive symmetric erythrokeratoderma (PSEK). To establish a causative relationship between loricrin mutations and these diseases, we have generated transgenic mice expressing a COOH-terminal truncated form of loricrin that is similar to the protein expressed in VS and PSEK patients. At birth, transgenic mice (ML.VS) exhibited erythrokeratoderma with an epidermal barrier dysfunction. 4 d after birth, high-expressing transgenic animals showed a generalized scaling of the skin, as well as a constricting band encircling the tail and, by day 7, a thickening of the footpads. Histologically, ML. VS transgenic mice also showed retention of nuclei in the stratum corneum, a characteristic feature of VS and PSEK. Immunofluorescence and immunoelectron microscopy showed the mutant loricrin protein in the nucleus and cytoplasm of epidermal keratinocytes, but did not detect the protein in the cornified cell envelope. Transfection experiments indicated that the COOH-terminal domain of the mutant loricrin contains a nuclear localization signal. To determine whether the ML.VS phenotype resulted from dominant-negative interference of the transgene with endogenous loricrin, we mated the ML.VS transgenics with loricrin knockout mice. A severe phenotype was observed in mice that lacked expression of wild-type loricrin. Since loricrin knockout mice are largely asymptomatic (Koch, P.K., P. A. de Viragh, E. Scharer, D. Bundman, M.A. Longley, J. Bickenbach, Y. Kawachi, Y. Suga, Z. Zhou, M. Huber, et al., J. Cell Biol. 151:389-400, this issue), this phenotype may be attributed to expression of the mutant form of loricrin. Thus, deposition of the mutant protein in the nucleus appears to interfere with late stages of epidermal differentiation, resulting in a VS-like phenotype.