Investigating Risk Factors for Urine Culture Contamination in Outpatient Clinics: A New Avenue for Diagnostic Stewardship.

Mixed flora in urine cultures usually occur due to pre-analytic contamination. In our outpatient urology clinic, we found a high prevalence of mixed flora (46.2%), which was associated with female sex and older age. Patient education did not impact the rate of mixed flora. Future efforts should target high-risk patients.

Small Diameter Penile Implants: A Survey on Current Utilization and Review of Literature.

BACKGROUND: Inflatable penile prostheses (IPPs) with smaller diameter cylinders have been in use for over 30 years, yet the literature is sparse on their utilization patterns amongst prosthetic surgeons. AIM: To understand current usage of small diameter penile implants (SDPI) among prosthetic surgeons. METHODS: IRB approval was obtained to conduct a survey of prosthetic surgeons. A 23-question online survey was distributed via email to physician members of the Sexual Medicine Society of North America (SMSNA) and Society of Urologic Prosthesis Surgeons (SUPS). The survey included questions regarding surgeon experience and volume, frequency of SDPI utilization, indications for SDPI, surgical strategy in the setting of SDPI (approach, use of concordant modeling/grafting), reservoir and pump management, and perceived infection risk and patient satisfaction. MAIN OUTCOME MEASURE: SDPI were utilized by the vast majority of respondents in certain clinical situations such as corporal fibrosis or anatomically small corpora, and surgeons have had a favorable experience with these as a final destination implant or as a place-holder until reimplantation with a normal diameter device. RESULTS: Fifty individuals responded to the survey, 48 of whom routinely utilized SDPI. The most common indication for SDPI placement was corporal fibrosis from prior infection, followed by anatomically small corpora and priapism. The most common maximal dilation diameter was 10 mm (47%), an additional 23% of respondents utilized SDPI with 11 mm dilation. 75.4% of respondents sometimes or always intended to upsize to standard diameter cylinders in the future. 68.8% of surgeons routinely counseled patients on the possibility of reduced grith and rigidity with SDPI. Patient satisfaction was perceived to be comparable to standard diameter cylinders in 56.3% of respondents, while the remaining 43.6% believed it to be lower than traditional cylinders. Utilization of SDPI can be an important tool for prosthetic surgeons faced with difficult cases due to corporal fibrosis or small corpora. This survey provides new insight into patterns of SDPI utilization by surgeons. A limitation of the study is that patient satisfaction is indirectly addressed through surgeons' perception and experience, further research will be necessary to include patient questionnaires regarding device satisfaction. CONCLUSION: SDPI are necessary in certain scenarios that preclude the use of normal diameter cylinders. These implants may offer satisfactory erections, but can also be upsized to standard diameter cylinders in the future. Campbell SP, Kim CJ, Allkanjari A et al. Small Diameter Penile Implants: A Survey on Current Utilization and Review of Literature. Sex Med 2022;10:100458.

Update on the Medical and Surgical Management of Urethral Condyloma.

INTRODUCTION: Condyloma acuminata (CA) of the urethra presents a management challenge due to high recurrence rates, difficulty in accessing urethral lesions, risk of stricture formation, and potential for sexual dysfunction. While standard treatment modalities are acceptable for some external genital condyloma, they are not always feasible or appropriate for urethral lesions. OBJECTIVES: We sought to review the literature on epidemiology, presentation, diagnosis and treatment of urothelial CA with a focus on surgical treatment options. METHODS: We performed a comprehensive literature search of PubMed to identify all studies pertaining to urethral CA through November 2020. RESULTS: Urethral CA is a relatively rare, but challenging disease to manage with a considerable amount of treatment side effects and downstream morbidity associated. In our comprehensive review we have found a wide selection of treatment modalities ranging from minimally invasive strategies to surgical reconstructive techniques. Proper follow-up to monitor for disease recurrence at the 3-4 month mark is appropriate and will determine subsequent treatment strategies as needed. Future studies and treatment directions include novel drug delivery models to optimize minimally invasive topical drug efficacy. CONCLUSION: Treatment of urethral CA should be approached in a step-wise fashion. Medical therapy would be an appropriate option for asymptomatic or minimally symptomatic patients with small lesions who desire to avoid any interventions. If patient is symptomatic, has extensive disease burden or has failed medical therapy intervention should be considered with options including PDT, laser ablation or surgical excision with or without urethral reconstruction. Appropriate selection depends on patient characteristics and preferences along with prior treatment history. Kim CJ, Campbell SP, Allkanjari A, et al. Update on the Medical and Surgical Management of Urethral Condyloma. Sex Med Rev 2022;238-252.

The exchange of the fast substrate water in the S2 state of photosystem II is limited by diffusion of bulk water through channels - implications for the water oxidation mechanism.

The molecular oxygen we breathe is produced from water-derived oxygen species bound to the Mn4CaO5 cluster in photosystem II (PSII). Present research points to the central oxo-bridge O5 as the 'slow exchanging substrate water (Ws)', while, in the S2 state, the terminal water ligands W2 and W3 are both discussed as the 'fast exchanging substrate water (Wf)'. A critical point for the assignment of Wf is whether or not its exchange with bulk water is limited by barriers in the channels leading to the Mn4CaO5 cluster. In this study, we measured the rates of H2 16O/H2 18O substrate water exchange in the S2 and S3 states of PSII core complexes from wild-type (WT) Synechocystis sp. PCC 6803, and from two mutants, D1-D61A and D1-E189Q, that are expected to alter water access via the Cl1/O4 channels and the O1 channel, respectively. We found that the exchange rates of Wf and Ws were unaffected by the E189Q mutation (O1 channel), but strongly perturbed by the D61A mutation (Cl1/O4 channel). It is concluded that all channels have restrictions limiting the isotopic equilibration of the inner water pool near the Mn4CaO5 cluster, and that D61 participates in one such barrier. In the D61A mutant this barrier is lowered so that Wf exchange occurs more rapidly. This finding removes the main argument against Ca-bound W3 as fast substrate water in the S2 state, namely the indifference of the rate of Wf exchange towards Ca/Sr substitution.

Roles of D1-Glu189 and D1-Glu329 in O2 Formation by the Water-Splitting Mn4Ca Cluster in Photosystem II.

During the catalytic step that precedes O-O bond formation in Photosystem II, a water molecule deprotonates and moves next to the water-splitting Mn4Ca cluster's O5 oxo bridge. The relocated oxygen, known as O6 or Ox, may serve as a substrate, combining with O5 to form O2 during the final step in the catalytic cycle, or may be positioned to become a substrate during the next catalytic cycle. Recent serial femtosecond X-ray crystallographic studies show that the flexibility of D1-E189 plays a critical role in facilitating the relocation of O6/Ox. In this study, the D1-E189G and D1-E189S mutations were characterized with FTIR difference spectroscopy. The data show that both mutations support Mn4Ca cluster assembly, substantially inhibit advancement beyond the S2 state, and alter the network of H bonds that surrounds the Mn4Ca cluster. Previously, the D1-E189Q, D1-E189K, and D1-E189R mutations were shown to have little impact on the activity, electron transfer rates, or spectral properties of Photosystem II. A rationale for this behavior is presented. The residue D1-E329 interacts with water molecules in the O1 water network that has been suggested recently to supply substrate during the catalytic cycle. Characterization of the D1-E329A mutant with FTIR difference spectroscopy shows that this mutation does not substantially perturb the structure of PSII or the water molecules whose O-H stretching modes change during the catalytic cycle. This result provides additional evidence that the water molecules whose vibrational properties change during the S1 to S2 transition are confined approximately to the region bounded by D1-N87, D1-N298, and D2-K317.

D1-S169A substitution of photosystem II reveals a novel S2-state structure.

In photosystem II (PSII), photosynthetic water oxidation occurs at the O2-evolving complex (OEC), a tetramanganese-calcium cluster that cycles through light-induced redox intermediates (S0-S4) to produce oxygen from two substrate water molecules. The OEC is surrounded by a hydrogen-bonded network of amino-acid residues that plays a crucial role in proton transfer and substrate water delivery. Previously, we found that D1-S169 was crucial for water oxidation and its mutation to alanine perturbed the hydrogen-bonding network. In this study, we demonstrate that the activation energy for the S2 to S1 transition of D1-S169A PSII is higher than wild-type PSII with a ~1.7-2.7× slower rate of charge recombination with QA- relative to wild-type PSII. Arrhenius analysis of the decay kinetics shows an Ea of 5.87 ± 1.15 kcal mol-1 for decay back to the S1 state, compared to 0.80 ± 0.13 kcal mol-1 for the wild-type S2 state. In addition, we find that ammonia does not affect the S2-state EPR signal, indicating that ammonia does not bind to the Mn cluster in D1-S169A PSII. Finally, a QM/MM analysis indicates that an additional water molecule binds to the Mn4 ion in place of an oxo ligand O5 in the S2 state of D1-S169A PSII. The altered S2 state of D1-S169A PSII provides insight into the S2➔S3 state transition.

Immunostaining Patterns of Posttransplant Liver Biopsies Using 2 Anti-C4d Antibodies.

Histopathologic diagnosis of antibody-mediated rejection in posttransplant liver biopsies is challenging. The recently proposed diagnostic criteria by the Banff Working Group on Liver Allograft Pathology require positive C4d immunohistochemical staining to establish the diagnosis. However, the reported C4d staining patterns vary widely in different studies. One potential explanation may be due to different antibody preparations used by different investigators. In this study, posttransplant liver biopsies from 69 patients histopathologically diagnosed with acute cellular rejection, chronic rejection, or recurrent hepatitis C were immunohistochemically stained using 2 polyclonal anti-C4d antibodies. On the basis of the distribution of C4d immunoreactivity, 5 different staining patterns were observed: portal vein and capillary, hepatic artery, portal stroma, central vein, and sinusoids. The frequency, extent, and intensity of positive C4d staining with the 2 antibody preparations differed significantly for portal veins/capillaries and central veins, but not for hepatic arteries and portal stroma. Positive sinusoidal staining was seen in only 1 case. There were no significant differences in the frequency, extent, and intensity of positive C4d staining among the acute cellular rejection, chronic rejection, and recurrent hepatitis C groups with the 2 anti-C4d antibodies. These data show that different anti-C4d antibodies can show different staining patterns, which may lead to different interpretation. Caution is thus needed when selecting C4d antibodies for clinical use to aid in the diagnosis of antibody-mediated rejection.

One of the Substrate Waters for O2 Formation in Photosystem II Is Provided by the Water-Splitting Mn4CaO5 Cluster's Ca2+ Ion.

During the catalytic step immediately prior to O-O bond formation in Photosystem II, a water molecule deprotonates and moves next to the water-splitting Mn4CaO5 cluster's O5 oxo bridge. Considerable evidence identifies O5 as one of the two substrate waters that ultimately form O2. The relocated oxygen, known as O6 or Ox, may be the second. It is currently debated whether O6 or Ox originates as the Mn-bound water denoted W2 or as the Ca2+-bound water denoted W3. To distinguish between these two possibilities, we analyzed the D-O-D bending mode of the water molecule that deprotonates/relocates to become O6/Ox. We show that this D-O-D bending mode is not altered by the D1-S169A mutation. Previously, we showed that this D-O-D bending mode is altered substantially when Sr2+ is substituted for Ca2+. Because Sr2+/Ca2+ substitution alters this D-O-D bending mode but the D1-S169A mutation does not, we conclude that the water-derived oxygen that relocates and becomes O6/Ox derives from the Ca2+-bound W3. This conclusion provides an important constraint for proposed mechanisms of O-O bond formation in Photosystem II.

Bicarbonate rescues damaged proton-transfer pathway in photosystem II.

The membrane-protein complex photosystem II (PSII) catalyzes photosynthetic water oxidation. Proton transfer plays an integral role in the catalytic cycle of water oxidation by maintaining charge balance to regulate and ensure the efficiency of the process. The hydrogen-bonded amino-acid residues that surround the oxygen-evolving complex (OEC) provide an efficient pathway for proton removal. Hence, it is crucial to identify these pathways to provide deeper insights into the proton-transfer mechanisms. In this study, we have used bicarbonate as a mobile exogenous proton-transfer reagent to recover the activity lost by site-directed mutations in order to identify amino-acid residues participating in the proton-transfer pathway. We find that bicarbonate restores efficient S-state cycling in D2-K317A PSII core complexes, but not in D1-D61A and CP43-R357K PSII core complexes, indicating that bicarbonate chemical rescue can be used to differentiate single-point mutations affecting the pathways of proton transfer from mutations that affect other aspects of the water-oxidation mechanism.

D1-S169A Substitution of Photosystem II Perturbs Water Oxidation.

In photosystem II (PSII), photosynthetic water oxidation occurs at the tetramanganese-calcium cluster that cycles through light-induced intermediates (S0-S4) to produce oxygen from two substrate waters. The surrounding hydrogen-bonded amino acid residues and waters form channels that facilitate proton transfer and substrate water delivery, thereby ensuring efficient water oxidation. The residue D1-S169 lies in the "narrow" channel and forms hydrogen bonds with the Mn4CaO5 cluster via waters W1 and Wx. To probe the role of the narrow channel in substrate-water binding, we studied the D1-S169A mutation. PSII core complexes isolated from mutant cells exhibit inefficient S-state cycling and delayed oxygen evolution. The S2-state multiline EPR spectrum of D1-S169A PSII core complexes differed significantly from that of wild-type, and FTIR difference spectra showed that the mutation strongly perturbs the extensive network of hydrogen bonds that extends at least from D1-Y161 (YZ) to D1-D61. These results imply a possible role of D1-S169 in proton egress or substrate water delivery.

Multiple Myeloma 1 Transcription Factor Is Superior to CD138 as a Marker of Plasma Cells in Endometrium.

Chronic endometritis is characterized by plasma cell (PC) infiltration of endometrial stroma. Identification of PCs can be challenging by routine hematoxylin and eosin (H&E) stain due to the low numbers of PCs or to their being obscured by other cells in the stroma. CD138 is widely used as an ancillary immunohistochemistry stain to identify PCs; however, it has a high background reaction. In this study, multiple myeloma 1 (MUM1) transcription factor is introduced as an alternative PC marker in endometrial tissues. In this study, 311 endometrial biopsies, submitted to rule out chronic endometritis, were selected. They were divided into Group I (n = 87) and Group II (n = 224). Both had MUM1 and H&E while Group I also had accompanying CD138 stains. In both groups combined, MUM1 detected plasma cells in 48% of the cases, while CD138 and H&E identified the cells in 23% and 15% of the biopsies, respectively. In addition to having a clean background, MUM1 is a more sensitive stain than CD138 for detection of PCs in endometrium.

Impact of D1-V185 on the Water Molecules That Facilitate O2 Formation by the Catalytic Mn4CaO5 Cluster in Photosystem II.

The oxidations of the O2-evolving Mn4CaO5 cluster in Photosystem II are coupled to the release of protons to the thylakoid lumen via one or more proton egress pathways. These pathways are comprised of extensive networks of hydrogen-bonded water molecules and amino acid side chains. The hydrophobic residue, D1-V185, is adjacent to numerous water molecules in one of these pathways. The D1-V185N mutation dramatically slows O-O bond formation. This impairment has been attributed to a disruption of the hydrogen-bonded water molecules that are crucial for proton egress or whose rearrangement is required for catalysis. In this study, Fourier transform infrared spectroscopy was employed to characterize the impact of the D1-V185N mutation on the carboxylate groups and water molecules that form a network of hydrogen bonds in this putative proton egress pathway. By analyzing carboxylate stretching modes, carbonyl stretching modes of hydrogen-bonded carboxylic acids, O-H stretching modes of hydrogen-bonded water molecules, and D-O-D bending modes, we obtain evidence that the D1-V185N mutation perturbs the extensive network of hydrogen bonds that extends from YZ to D1-D61 to a greater extent than any mutation yet examined but does not alter the water molecules that interact directly with D1-D61. The mutation also alters the environments of the carboxylate groups whose p Ka values change in response to the S1 to S2 and S2 to S3 transitions. Finally, the mutation alters the environment of the water molecule whose bending mode vanishes during the S2 to S3 transition, consistent with assigning the Ca2+-bound W3 as the water molecule that deprotonates and joins oxo bridge O5 during the S2 to S3 transition, possibly as the second substrate water molecule for O2 formation.

Substitution of the D1-Asn87 site in photosystem II of cyanobacteria mimics the chloride-binding characteristics of spinach photosystem II.

Photoinduced water oxidation at the O2-evolving complex (OEC) of photosystem II (PSII) is a complex process involving a tetramanganese-calcium cluster that is surrounded by a hydrogen-bonded network of water molecules, chloride ions, and amino acid residues. Although the structure of the OEC has remained conserved over eons of evolution, significant differences in the chloride-binding characteristics exist between cyanobacteria and higher plants. An analysis of amino acid residues in and around the OEC has identified residue 87 in the D1 subunit as the only significant difference between PSII in cyanobacteria and higher plants. We substituted the D1-Asn87 residue in the cyanobacterium Synechocystis sp. PCC 6803 (wildtype) with alanine, present in higher plants, or with aspartic acid. We studied PSII core complexes purified from D1-N87A and D1-N87D variant strains to probe the function of the D1-Asn87 residue in the water-oxidation mechanism. EPR spectra of the S2 state and flash-induced FTIR spectra of both D1-N87A and D1-N87D PSII core complexes exhibited characteristics similar to those of wildtype Synechocystis PSII core complexes. However, flash-induced O2-evolution studies revealed a decreased cycling efficiency of the D1-N87D variant, whereas the cycling efficiency of the D1-N87A PSII variant was similar to that of wildtype PSII. Steady-state O2-evolution activity assays revealed that substitution of the D1 residue at position 87 with alanine perturbs the chloride-binding site in the proton-exit channel. These findings provide new insight into the role of the D1-Asn87 site in the water-oxidation mechanism and explain the difference in the chloride-binding properties of cyanobacterial and higher-plant PSII.

Androgen deprivation therapy for the treatment of prostate cancer: a focus on pharmacokinetics.

INTRODUCTION: Medical therapy has undergone many changes as our understanding of prostate cancer cell biology has improved. Androgen deprivation therapy (ADT) remains the mainstay of therapy for metastatic disease. Metastatic castrate-resistant prostate cancer (CRPC) is an important concern since we are unable to stop progression with currently available agents. Areas covered: Pharmacologic ADT is the most commonly used treatment for metastatic prostate cancer. Multiple agents are available for both first-line and second-line use: antiandrogens, estrogens, luteinizing hormone-releasing hormone agonists/antagonists, and CYP17 inhibitors. With adoption of these drugs, it is important to consider their pharmacokinetic and pharmacodynamic properties. Many undergo metabolism through cytochrome P450. Levels may be altered with co-administration of drugs acting as enzyme inhibitors or inducers. Understanding mechanism of action, metabolism, and excretion of these drugs allows clinicians to provide the best therapeutic care while minimizing adverse events. Expert opinion: Many men with metastatic prostate cancer will progress to castration resistance. An understanding of resistance mechanisms at the cellular level has revealed new drug targets with hopes of halting or reversing progression of metastatic disease. Second-line agents, traditionally reserved for CRPC, are being studied in metastatic castrate-sensitive prostate cancer, and may offer practice-changing evidence supporting their use.

Renal cell carcinoma: the search for a reliable biomarker.

One particular challenge in the treatment of kidney tumors is the range of histologies and tumor phenotypes a renal mass can represent. A kidney tumor can range from benign (e.g., oncocytoma) to a clinically indolent malignancy (e.g., papillary type I, chromophobe) to aggressive disease [e.g., papillary type II or high-grade clear cell renal cell carcinoma (ccRCC)]. Even among various subtypes, kidney cancers are genetically diverse with variable prognoses and treatment response rates. Therefore, the key to proper treatment is the differentiation of these subtypes. Currently, a wide array of diagnostic, prognostic, and predictive biomarkers exist that may help guide the individualized care of kidney cancer patients. This review will discuss the various serum, urine, imaging, and immunohistological biomarkers available in practice.

Practical Immunohistochemistry in Neoplastic Pathology of the Gastrointestinal Tract, Liver, Biliary Tract, and Pancreas.

CONTEXT: - Immunomarkers with diagnostic, therapeutic, or prognostic values have been increasingly used to maximize the benefits of clinical management of patients with neoplastic diseases of the gastrointestinal tract, liver, biliary tract, and pancreas. OBJECTIVES: - To review the characteristics of immunomarkers that are commonly used in surgical pathology practice for neoplasms of the gastrointestinal tract, liver, biliary tract, and pancreas, and to summarize the clinical usefulness of immunomarkers that have been discovered in recent years in these fields. DATA SOURCES: - Data sources include literature review, authors' research data, and personal practice experience. CONCLUSIONS: - Immunohistochemistry is an indispensable tool for the accurate diagnosis of neoplastic diseases of the gastrointestinal tract, liver, biliary tract, and pancreas. Useful immunomarkers are available to help distinguish malignant neoplasms from benign conditions, determine organ origins, and subclassify neoplasms that are morphologically and biologically heterogeneous. Specific immunomarkers are also available to help guide patient treatment and assess disease aggressiveness, which are keys to the success of personalized medicine. Pathologists will continue to play a critical role in the discovery, validation, and application of new biomarkers, which will ultimately improve patient care.

Evidence from FTIR Difference Spectroscopy That a Substrate H2O Molecule for O2 Formation in Photosystem II Is Provided by the Ca Ion of the Catalytic Mn4CaO5 Cluster.

The O2-producing Mn4CaO5 catalyst in photosystem II oxidizes two water molecules (substrate) to produce one O2 molecule. Considerable evidence supports the identification of one of the two substrate waters as the Mn4CaO5 cluster's oxo bridge known as O5. The identity of the second substrate water molecule is less clear. In one set of models, the second substrate is the Mn-bound water molecule known as W2. In another set of models, the second substrate is the Ca2+-bound water molecule known as W3. In all of these models, a deprotonated form of the second substrate moves to a position next to O5 during the catalytic step immediately prior to O-O bond formation. In this study, FTIR difference spectroscopy was employed to identify the vibrational modes of hydrogen-bonded water molecules that are altered by the substitution of Sr2+ for Ca2+. Our data show that the substitution substantially altered the vibrational modes of only a single water molecule: the water molecule whose D-O-D bending mode is eliminated during the catalytic step immediately prior to O-O bond formation. These data are most consistent with the identification of the Ca2+-bound W3 as the second substrate involved in O-O bond formation.

Value of SATB2 immunostaining in the distinction between small intestinal and colorectal adenocarcinomas.

AIMS: Special AT-rich sequence-binding protein 2 (SATB2) is a novel immunomarker that is expressed in glandular cells of the lower gastrointestinal tract with retained expression in the majority of primary and metastatic colorectal adenocarcinomas (CRCs). Because of its tissue specificity, SATB2 has been shown to be a clinically useful marker to distinguish CRC from non-CRC. In this study, we investigated whether or not SATB2 can help differentiate CRC from small intestinal adenocarcinoma (SIA), a practical diagnostic challenge due to their morphological and immunophenotypic similarities. METHODS: Fifty surgically resected primary SIAs and 50 CRCs were immunohistochemically examined for the expression of SATB2. Positive staining was graded as 1+ (5-25% of the tumour cells stained), 2+ (26-50%), 3+ (51-75%) or 4+ (>75%), as well as weak, intermediate or strong for staining intensity. RESULTS: Positive SATB2 immunoreactivity was observed in 23 (46%) SIAs in contrast to 48 (96%) CRCs (p<0.0001). Among these, only 4 (8%) SIAs showed strong and diffuse (4+) SATB2 staining compared with 38 (76%) of CRCs (p<0.0001). CONCLUSIONS: SATB2 is not entirely CRC-specific and is expressed in a subset of SIAs. Unlike CRC, however, SIA infrequently shows a strong and diffuse staining pattern, which still makes SATB2 a useful immunomarker to distinguish SIA from CRC.

Gemcitabine Plus Radiation Therapy for High-Grade Glioma: Long-Term Results of a Phase 1 Dose-Escalation Study.

PURPOSE: To evaluate the tolerability and efficacy of gemcitabine plus radiation therapy (RT) in this phase 1 study of patients with newly diagnosed malignant glioma (HGG). PATIENTS AND METHODS: Between 2004 and 2012, 29 adults with HGG were enrolled. After any extent of resection, RT (60 Gy over 6 weeks) was given concurrent with escalating doses of weekly gemcitabine. Using a time-to-event continual reassessment method, 5 dose levels were evaluated starting at 500 mg/m(2) during the last 2 weeks of RT and advanced stepwise into earlier weeks. The primary objective was to determine the recommended phase 2 dose of gemcitabine plus RT. Secondary objectives included progression-free survival, overall survival (OS), and long-term toxicity. RESULTS: Median follow-up was 38.1 months (range, 8.9-117.5 months); 24 patients were evaluable for toxicity. After 2005 when standard practice changed, patients with World Health Organization grade 4 tumors were no longer enrolled. Median progression-free survival for 22 patients with grade 3 tumors was 26.0 months (95% confidence interval [CI] 15.6-inestimable), and OS was 48.5 months (95% CI 26.8-inestimable). In 4 IDH mutated, 1p/19q codeleted patients, no failures occurred, with all but 1 alive at time of last follow-up. Seven with IDH mutated, non-codeleted tumors with ATRX loss had intermediate OS of 73.5 months (95% CI 32.8-inestimable). Six nonmutated, non-codeleted patients had a median OS of 26.5 months (95% CI 25.4-inestimable). The recommended phase 2 dose of gemcitabine plus RT was 750 mg/m(2)/wk given the last 4 weeks of RT. Dose reductions were most commonly due to grade 3 neutropenia; no grade 4 or 5 toxicities were seen. CONCLUSIONS: Gemcitabine concurrent with RT is well-tolerated and yields promising outcomes, including in patients with adverse molecular features. It is a candidate for further study, particularly for poor-prognosis patient subgroups with HGG.

An adapter ligation-mediated PCR method for high-throughput mapping of T-DNA inserts in the Arabidopsis genome.

Agrobacterium transfer DNA (T-DNA) is an effective plant mutagen that has been used to create sequence-indexed T-DNA insertion lines in Arabidopsis thaliana as a tool to study gene function. Creating T-DNA insertion lines requires a dependable method for locating the site of insertion in the genome. In this protocol, we describe an adapter ligation-mediated PCR method that we have used to screen a mutant library and identify over 150,000 T-DNA insertional mutants; the method can also be applied to map individual mutants. The procedure consists of three steps: a restriction enzyme-mediated ligation of an adapter to the genomic DNA; a PCR amplification of the T-DNA/genomic DNA junction with primers specific to the adapter and T-DNA; and sequencing of the T-DNA/genomic junction to enable mapping to the reference genome. In most cases, the sequenced genomic region extends to the T-DNA border, enabling the exact location of the insert to be identified. The entire process takes 2 weeks to complete.