Harmful algal blooms in Cayuga lake, NY: From microbiome analysis to eDNA monitoring.

The global increase in harmful algal blooms (HABs) has become a growing concern over the years, and New York State (NYS) is no exception. The Finger Lakes region in NYS has been identified as a hotspot for HABs, with Cayuga Lake having the highest number of blooms reported. The Cayuga Lake HABs Monitoring Program has been tracking cHABs (dominant bloom taxa, chlorophyll A, and microcystin levels) since 2018. However, limited research has been conducted on the microbiome of HABs in this region. In this study, the microbiome of HABs in the Cayuga Lake was surveyed and compared with non-HAB baseline samples. Using 16S rDNA community analysis, common bloom-forming cyanobacteria, were identified, with Microcystis being the dominant taxa in high toxin blooms. Further, this study evaluated the ability of Microcystis mcyA qPCR to detect elevated levels of potential toxigenic Microcystis in water samples using both benchtop and handheld qPCR devices. The results showed good performance of the qPCR assay as a screening for high toxin versus low/no toxin blooms. Additionally, the handheld qPCR device holds potential for in-field rapid (<1 h) screenings for high toxin blooms. This study provides insights into the microbiome of HABs in Cayuga Lake and offers a potential tool for rapid screening of high toxin blooms.

Tuning Azoheteroarene Photoswitch Performance through Heteroaryl Design.

Photoswitchable compounds, which can be reversibly switched between two isomers by light, continue to attract significant attention for a wide array of applications. Azoheteroarenes represent a relatively new but understudied type of photoswitch, where one of the aryl rings from the conventional azobenzene class has been replaced with a five-membered heteroaromatic ring. Initial studies have suggested the azoheteroarenes-the arylazopyrazoles in particular-to have excellent photoswitching properties (quantitative switching and long Z isomer half-life). Here we present a systematic computational and experimental study to elucidate the origin of the long thermal half-lives and excellent addressability of the arylazopyrazoles, and apply this understanding to determine important structure-property relationships for a wide array of comparable azoheteroaryl photoswitches. We identify compounds with Z isomer half-lives ranging from seconds to hours, to days and to years, and variable absorption characteristics, all through tuning of the heteraromatic ring. Conformation perhaps plays the largest role in determining such properties: the compounds with the longest isomerization half-lives adopt a T-shaped ground state Z isomer conformation and proceed through a T-shaped isomerization pathway, whereas the most complete photoswitching is achieved for compounds that have a twisted (rather than T-shaped) Z isomer conformation. By balancing these factors, we report a new azopyrazole 3pzH, which can be quantitatively switched to its Z isomer (>98%) with 355 nm irradiation, near-quantitatively (97%) switched back to the E isomer with 532 nm irradiation, and has a very long half-life for thermal isomerization (t1/2 = 74 d at 25 °C). Given the large tunability of their properties, the predictive nature of their performance, and the other functional opportunities afforded by usage of a heteroaromatic system, we believe the azoheteroaryl photoswitches to have huge potential in a wide range of optically addressable applications.

Arylazopyrazoles: azoheteroarene photoswitches offering quantitative isomerization and long thermal half-lives.

Arylazopyrazoles, a novel class of five-membered azo photoswitches, offer quantitative photoswitching and high thermal stability of the Z isomer (half-lives of 10 and ∼1000 days). The conformation of the Z isomers of these compounds, and also the arylazopyrroles, is highly dependent on the substitution pattern on the heteroarene, allowing a twisted or planar geometry, which in turn has a significant impact on the electronic spectral properties of the compounds.

The JNK signal transduction pathway.

The c-Jun NH(2)-terminal kinases (JNKs) are an evolutionarily conserved sub-group of mitogen-activated protein (MAP) kinases. Recent studies have improved our understanding of the physiological function of the JNK pathway. Roles of novel molecules that participate in the JNK pathway have been defined and new insight into the role of JNK in survival signaling, cell death, cancer and diabetes has been achieved.

Delay between symptom onset and clinic attendance following TIA and minor stroke: the BEATS study.

BACKGROUND: rapid specialist assessment of patients with transient ischaemic attack (TIA) reduces the risk of recurrent stroke. National guidelines advise that high-risk patients are assessed within 24 h and low-risk patients within 7 days. AIM: to quantify delay and map pathways taken by patients from symptom onset to specialist assessment. DESIGN: retrospective cohort study. SETTING: rapid access TIA clinic. METHODS: structured interviews with 278 patients newly diagnosed with TIA (222) or minor stroke (56), and examination of medical records. RESULTS: of the 133 high-risk TIA patients, 11 (8%) attended the clinic within 24 h of symptom onset; of the 89 low-risk TIA patients, 47 (53%) attended within 7 days. Median delay between symptom onset and seeking help from a healthcare professional (HCP) was 4.0 h (IQR 0.5, 41.3). Delay was less if symptoms were correctly interpreted but not reduced by a publicity campaign (FAST) to encourage an urgent response. Most patients (156, 56%) first contacted a general practitioner (GP) and 46 (17%) called an ambulance or attended the emergency department. Over a third (36%) had a second consultation with an HCP before attending the clinic, and this was more likely in those presenting to paramedics, out of hours GP services or optometry. Time to clinic attendance was less if an emergency pathway was used and greater if patients were seen by a second HCP. CONCLUSIONS: factors contributing to delay include incorrect interpretation of symptoms and failure to invoke emergency services. Delays after presentation could be addressed by direct referral by out of hours services, paramedics and optometrists.

Exploring Reasons for Non-Engagement From a Peer-Led Diversionary Intervention for Veterans in Police Custody.

UK veterans with complex needs arrested in police custody can access support through pre-charge diversion into treatment and ancillary services. We consider why veterans in police custody disengaged from a peer-led criminal justice diversionary support service in one UK region that adopted a continuous case management approach. Seven hundred and fifty-seven veterans were assessed to have high levels of comorbid health needs and socio-economic harms, with one-quarter (26.7%, n = 202) subsequently disengaging from the service. A logistic regression model using Multivariate Imputation by Chained Equations identified that veterans of a younger age, no-fixed-abode, a history of incarceration, and those from a Royal Navy background were likelier to disengage from the intervention. We conclude that this peer-based diversionary model has some efficacy in maintaining the engagement of a highly complex, comorbid segment of criminally-justice-exposed UK military veterans. The perceived benefits of an integrated peer-based model predicated on continuous case-management techniques are discussed.

Primary follicular lymphoma of the testis in children and adolescents.

This study reports 6 cases of primary follicular lymphoma of the testis (PFLT) in children and adolescents correlated with clinical presentation, pathologic features, treatment, and outcome. All 6 patients (age, 3 to 16 y; median, 4 y) had PFLT grade 3 with disease limited to the testis, completely resected and treated with 2 courses of chemotherapy (cyclophosphamide, vincristine, prednisone, doxorubicin). Event-free survival was 100% (follow-up: median, 73 mo; mean, 53 mo; range, 6 to 96 mo). In conclusion, clinical outcome in children and adolescents with PFLT is excellent with treatment including complete surgical resection and 2 courses of cyclophosphamide, vincristine, prednisone, doxorubicin.

Excellent survival following two courses of COPAD chemotherapy in children and adolescents with resected localized B-cell non-Hodgkin's lymphoma: results of the FAB/LMB 96 international study.

High cure rates are possible in children with localized mature B-cell lymphoma (B NHL) using a variety of chemotherapeutic strategies. To reduce late sequelae, the duration and intensity of chemotherapy has been progressively reduced. The Lymphome Malins de Burkitt (LMB) 89 study reported long-term survival in almost all children with localized resected disease treated with two courses of COPAD (cyclophosphamide, vincristine, prednisolone and doxorubicin). This study was designed to confirm the effectiveness of this approach in a larger number of patients in a multinational co-operative study. The patient cohort was part of an international study (French-American-British LMB 96), which included all disease stages and involved three national groups. Patients in this part of the study had resected stage I or completely resected abdominal stage II disease. Following surgery, two courses of COPAD were given, without intrathecal (IT) chemotherapy. One hundred and thirty-two children were evaluable. Two of 264 (0.9%) courses were associated with grade IV toxicity (one stomatitis and one infection). With a median follow up of 50.5 months, the 4 year event-free survival is 98.3% and overall survival is 99.2%. Children with resected localized B-NHL can be cured with minimal toxicity following two courses of low intensity treatment without IT chemotherapy.

The JNK signal transduction pathway.

The c-Jun NH(2)-terminal kinase (JNK) is a member of an evolutionarily conserved sub-family of mitogen-activated protein (MAP) kinases. Recent studies have led to progress towards understanding the physiological function of the JNK signaling pathway, including the analysis of the phenotype of knockout mice. An important role for JNK in the non-canonical Wnt-signaling pathway has been established. Insight into the role of scaffold proteins that may assemble functional JNK modules has been achieved. In addition, a small molecule pharmacological inhibitor of JNK has been described and it is likely that this drug will facilitate future studies of JNK function.

Nodule formation and desmoplasia in medulloblastomas-defining the nodular/desmoplastic variant and its biological behavior.

Among the variants of medulloblastoma in the current WHO classification of nervous system tumors, the desmoplastic variant, which has been reported to constitute 5%-25% of pediatric medulloblastomas, is defined by its nodular collections of neurocytic cells bounded by desmoplastic internodular zones. We have studied the frequency, morphological features and biological behavior of medulloblastomas in two contemporaneous SIOP/UKCCSG trial cohorts of children with medulloblastomas, CNS9102 (n = 315) and CNS9204 (n = 35), focusing on tumors with nodular and desmoplastic phenotypes. In children aged 3-16 years (CNS9102), the nodular/desmoplastic medulloblastoma represented 5% of all tumors, while in infants aged <3 years (CNS9204) this variant represented 57% of medulloblastomas. Using iFISH to detect molecular cytogenetic abnormalities in medulloblastomas with a nodular architecture, we demonstrated distinct genetic profiles in desmoplastic and non-desmoplastic (classic and anaplastic) tumors; in particular, abnormalities of chromosome 17 occurred in the latter, but not the former. Significantly different outcomes were demonstrated for classic, nodular/desmoplastic and large cell/anaplastic medulloblastomas in both cohorts. In conclusion, the nodular/desmoplastic medulloblastoma appears to have clinical, genetic and biological characteristics that set it apart from other variants of this tumor.

Long-term outcome in children with Hodgkin's lymphoma: the United Kingdom Children's Cancer Study Group HD82 trial.

BACKGROUND: The aim of United Kingdom Children's Cancer Study Group (UKCCSG) HD82 was to establish the efficacy of chlorambucil/vinblastine/procarbazine/prednisolone (ChlVPP) in the treatment of childhood Hodgkin's lymphoma stages II-IV and radiotherapy (RT) alone in stage I patients. We report on the status of these patients to a follow-up of 20 years. METHODS: Treatment consisted of 35Gy involved-field RT for stage I and ChlVPP alone for stages II-IV. Adjuvant RT (35Gy) was administered to those with bulky mediastinal disease. RESULTS: Of the 358 patients, the 10-year EFS/OS per stage is I (65.4%/92.6%), II (80.0%/93.3%), III (68.8%/85.0%), IV (45.5%/72.7%). The corresponding 20-year OS rates are similar with a combined (all stage) rate dropping from 89.3% to 89.0% over the decade. The cumulative 20-year malignancy rate is 7.29%. CONCLUSION: Single modality treatment provided relatively low EFS at 10-years but comparable long-term OS, relative to contemporary published combined modality regimens, for stages I-III but not for stage IV patients.

Physiotherapy postgraduate studies in South Africa: Facilitators and barriers.

AIM: To investigate the facilitators and barriers to attaining a postgraduate physiotherapy degree in South Africa. METHODS: A quantitative, cross-sectional design using an internet-based survey was employed. The population of the study included all qualified physiotherapists who had completed community service and who were on the South African Society of Physiotherapy e-mailing list at the time of the study. RESULTS: In all, 425 valid responses were received. The study participants were predominantly white women with a mean age of 36.9 and the majority were working in private practice. A total of 20.5% of respondents had completed a master's or doctoral degree in physiotherapy, while a further 13% of respondents were registered for a postgraduate degree in physiotherapy at the time of the study. Study participants who had obtained a postgraduate degree identified the same main barriers (namely cost/lack of financial support, family commitments and lack of time) and the same main facilitators (namely gaining of expertise, fulfilment of a personal goal and improvement of patient care) as participants who had not obtained a postgraduate degree. Participants who had not obtained a postgraduate degree were significantly more likely (p < 0.05) to report concerns regarding their own ability and a lack of motivation as barriers to further study. CONCLUSION: South African physiotherapists with and without a postgraduate degree reported common facilitators and barriers to pursuing postgraduate studies. In order to ensure that a greater number and diversity of physiotherapists see postgraduate studies as a worthwhile career option, stakeholders in health and education in both the South African public and private sectors need to be engaged to limit the barriers to postgraduate study and seek novel methods of making postgraduate study a more attractive option from a personal development and career perspective.

Toward Photopharmacological Antimicrobial Chemotherapy Using Photoswitchable Amidohydrolase Inhibitors.

Photopharmacological agents exhibit light-dependent biological activity and may have potential in the development of new antimicrobial agents/modalities. Amidohydrolase enzymes homologous to the well-known human histone deacetylases (HDACs) are present in bacteria, including resistant organisms responsible for a significant number of hospital-acquired infections and deaths. We report photopharmacological inhibitors of these enzymes, using two classes of photoswitches embedded in the inhibitor pharmacophore: azobenzenes and arylazopyrazoles. Although both classes of inhibitor show excellent inhibitory activity (nM IC50 values) of the target enzymes and promising differential activity of the switchable E- and Z-isomeric forms, the arylazopyrazoles exhibit better intrinsic photoswitch performance (more complete switching, longer thermal lifetime of the Z-isomer). We also report protein-ligand crystal structures of the E-isomers of both an azobenzene and an arylazopyrazole inhibitor, bound to bacterial histone deacetylase-like amidohydrolases (HDAHs). These structures not only uncover interactions important for inhibitor binding but also reveal conformational differences between the two photoswitch inhibitor classes. As such, our data may pave the way for the design of improved photopharmacological agents targeting the HDAC superfamily.

beta-Catenin status predicts a favorable outcome in childhood medulloblastoma: the United Kingdom Children's Cancer Study Group Brain Tumour Committee.

PURPOSE: Identifying pathobiological correlates of clinical behavior or therapeutic response currently represents a key challenge for medulloblastoma research. Nuclear accumulation of the beta-catenin protein is associated with activation of the Wnt/Wg signaling pathway, and mutations affecting components of this pathway have been reported in approximately 15% of sporadic medulloblastomas. We tested the hypothesis that nuclear immunoreactivity for beta-catenin is a prognostic marker in medulloblastoma, and assessed the relationship between nuclear beta-catenin immunoreactivity and mutations of CTNNB1 and APC. PATIENTS AND METHODS: Medulloblastomas from children entered onto the International Society for Pediatric Oncology (SIOP)/United Kingdom Children's Cancer Study Group (UKCCSG) PNET3 trial (n = 109) were examined for beta-catenin immunoreactivity, and where tissue was available, evidence of CTNNB1 and APC mutations. The results were correlated with clinicopathologic variables, principally outcome. RESULTS: Children with medulloblastomas that showed a nucleopositive beta-catenin immunophenotype (27 of 109; 25%) had significantly better overall (OS) and event-free (EFS) survivals than children with tumors that showed either membranous/cytoplasmic beta-catenin immunoreactivity or no immunoreactivity (P = .0015 and P = .0026, respectively). For beta-catenin nucleopositive and nucleonegative medulloblastomas, 5-year OS was 92.3% (95% CI, 82% to 100%) versus 65.3% (95% CI, 54.8 to 75.7%), and 5-year EFS was 88.9% (95% CI, 77% to 100%) versus 59.5% (95% CI, 48.8 to 70.2%), respectively. Mutations in CTNNB1 were found exclusively among medulloblastomas that demonstrated nuclear beta-catenin immunoreactivity, but no evidence of APC mutation was found in these cases. All children with beta-catenin nucleopositive large cell/anaplastic medulloblastomas and beta-catenin nucleopositive medulloblastomas presenting with metastatic disease are alive at least 5 years postdiagnosis. CONCLUSION: Nuclear accumulation of beta-catenin appears to be a marker of favorable outcome in medulloblastoma, and should be investigated further in large group-wide trials.

Analysis of patients with supratentorial primitive neuro-ectodermal tumours entered into the SIOP/UKCCSG PNET 3 study.

The SIOP PNET 3 study was designed to determine whether 10 weeks of moderately intensive chemotherapy given after surgery and before radiotherapy (RT) would improve the outcome for patients with primitive neuroectodermal tumours (PNETs) compared with RT alone. Patients with a histological diagnosis of supratentorial PNET (StPNET) and no radiological evidence of metastatic disease were initially eligible for randomisation to either chemotherapy followed by craniospinal RT 35 Gy in 21 fractions with a boost of 20 Gy in 12 fractions to the primary site, or RT alone. In respect of the increasing recognition that StPNET were high-risk tumours, randomisation for this group closed in November 1999. This analysis includes both randomised and non-randomised patients with StPNET entered into the study database. Sixty-eight patients aged 2.9-16.6 years (median 6.5 years) were included in the analysis (chemotherapy+RT: 44, RT alone: 24). Fifty-four patients (79%) had a non-pineal and 14 (21%) a pineal site. At a median follow-up of 7.4 years, for all patients overall survival (OS) at 3 and 5 years was 54.4% and 48.3%, respectively. Event-free survival (EFS) at 3 and 5 years was 50.0% and 47.0%, respectively. There was no statistically significant difference in OS or EFS according to treatment received. OS (P=0.05) and EFS (P=0.03) were significantly better for patients with pineal primary sites. EFS for pineal tumours were 92.9% at 3 years and 71.4% at 5 years and for non-pineal primaries 40.7% at 3 years and 40.7% at 5 years. This study confirmed the relatively good survival for non-metastatic pineal PNETs but poor survival of non-pineal StPNETs. There was no evidence that pre-radiation chemotherapy improved outlook. Future treatment programs should be directed at the particular natural history of these tumours, to further define prognostic factors and to explore further biological characteristics.

The conditional kinase DeltaMEKK1:ER* selectively activates the JNK pathway and protects against serum withdrawal-induced cell death.

The conditional protein kinase DeltaMEKK3:ER* allows activation of the mitogen-activated and stress-activated protein kinases (MAPKs and SAPKs) without imposing a primary cellular stress or damage. Such separation of stress from stress-induced signalling is particularly important in the analysis of apoptosis. Activation of DeltaMEKK3:ER* in cycling CCl39 cells caused a rapid stimulation of the ERK1/2, JNK and p38 pathways but resulted in a slow, delayed apoptotic response. Paradoxically, activation of the same pathways inhibited the rapid expression of Bim(EL) and apoptosis following withdrawal of serum. Inhibition of the ERK1/2 pathway prevented the down-regulation of Bim(EL) but caused only a partial reversion of the cyto-protective effect of DeltaMEKK3:ER*. In contrast, inhibition of p38 had no effect, raising the possibility that activation of JNK might also exert a protective effect. To test this we used CCl39 cells expressing DeltaMEKK1:ER* which activates JNK but not ERK1/2, p38, PKB or IkappaB kinase. Activation of DeltaMEKK1:ER* inhibited serum withdrawal-induced conformational changes in Bax and apoptosis. These results suggest that in the absence of any overt cellular damage or chemical stress activation of JNK can act independently of the ERK1/2 or PKB pathways to inhibit serum withdrawal-induced cell death.

Photoswitchable basicity through the use of azoheteroarenes.

Azoheteroarene photoswitches offer functional advantages over their more conventional azobenzene counterparts by virtue of their heteroaromatic ring(s). Here we report that azobis(2-imidazole) functions as a photoswitchable base due to the additional proton stabilisation that is possible in the protonated Z isomer, facilitated by the basic imidazole nitrogens. This thermodynamic difference in stability corresponds to a 1.3 unit difference in pK(a) values between the E and Z isomers. This pK(a) difference can be used to reversibly control solution pH.

Delta MEKK3:ER* activation induces a p38 alpha/beta 2-dependent cell cycle arrest at the G2 checkpoint.

Whilst many studies have examined the role of the MAP Kinases in regulating the G1-->S transition, much less is known about the function of these pathways in regulating other cell cycle transitions. Stimulation of the conditional mutant Delta MEKK3:ER* in asynchronous hamster (CCl39) and rat (Rat-1) fibroblasts resulted in the strong activation of endogenous JNK and p38 but only a weak activation of ERK. Activation of Delta MEKK3:ER* inhibited cell proliferation through a combination of an initial G1 and G2 cell cycle arrest, followed by a delayed onset of apoptosis. When cells were synchronized in S phase with aphidicolin and then released, activation of Delta MEKK3:ER* resulted in the up-regulation of p21(CIP1) and a pronounced inhibition of cyclin A/CDK2 and cyclin B1/CDK1 kinase activity. Analysis of mitotic figures indicated that cells failed to enter mitosis, arresting late in G2. Delta MEKK3:ER*-mediated CDK inhibition and G2 arrest did not absolutely require p21(CIP1), since both events were observed in Rat-1 cells in which p21(CIP1) is transcriptionally silenced due to promoter methylation. Rather, CDK inhibition was associated with a down-regulation of cyclin A and B1 expression. Finally, application of the p38 inhibitor SB203580 partially restored cyclin B associated kinase activity and allowed cells to proceed through mitosis into the next G1 phase, suggesting that activation of the p38 alpha/beta 2 pathway can promote a G2 cell cycle arrest.

ERK1/2 and p38 cooperate to induce a p21CIP1-dependent G1 cell cycle arrest.

To study the mechanisms by which mitogen- and stress-activated protein kinases regulate cell cycle re-entry, we have used a panel of conditional kinases that stimulate defined MAPK or SAPK cascades. Activation of DeltaMEKK3:ER* during serum restimulation of quiescent cells causes a strong activation of JNK1 and p38alpha but only a modest potentiation of serum-stimulated ERK1/2 activity. In CCl39 cells this promoted a sustained G1 arrest that correlated with decreased expression of cyclin D1 and Cdc25A, increased expression of p21CIP1 and inhibition of CDK2 activity. In Rat-1 cells, in which p21(CIP1) expression is silenced by methylation, DeltaMEKK3:ER* activation caused only a transient delay in the S phase entry rather than a sustained G1 arrest. Furthermore, p21CIP1-/- 3T3 cells were defective for the DeltaMEKK3:ER*-induced G1 cell cycle arrest compared to their wild-type counterparts. These results suggest that activated DeltaMEKK3:ER* inhibits the G1 --> S progression by two kinetically distinct mechanisms, with expression of p21CIP1 being required to ensure a sustained G1 cell cycle arrest. The ERK1/2 and p38alphabeta pathways cooperated to induce p21CIP1 expression and inhibition of p38alphabeta caused a partial reversal of the cell cycle arrest. In contrast, selective activation of ERK1/2 by DeltaRaf-1:ER* did not inhibit serum stimulated cell cycle re-entry. Finally, selective activation of JNK by DeltaMEKK1:ER* failed to inhibit cell cycle re-entry, even in cells that retained wild-type p53, arguing against a major role for JNK alone in antagonizing the G1 --> S transition.