CD4 T Cell-Derived IL-21 Is Critical for Sustaining Plasmodium Infection-Induced Germinal Center Responses and Promoting the Selection of Memory B Cells with Recall Potential.

Development of Plasmodium-specific humoral immunity is critically dependent on CD4 Th cell responses and germinal center (GC) reactions during blood-stage Plasmodium infection. IL-21, a cytokine primarily produced by CD4 T cells, is an essential regulator of affinity maturation, isotype class-switching, B cell differentiation, and maintenance of GC reactions in response to many infection and immunization models. In models of experimental malaria, mice deficient in IL-21 or its receptor IL-21R fail to develop memory B cell populations and are not protected against secondary infection. However, whether sustained IL-21 signaling in ongoing GCs is required for maintaining GC magnitude, organization, and output is unclear. In this study, we report that CD4+ Th cells maintain IL-21 expression after resolution of primary Plasmodium yoelii infection. We generated an inducible knockout mouse model that enabled cell type-specific and timed deletion of IL-21 in peripheral, mature CD4 T cells. We found that persistence of IL-21 signaling in active GCs had no impact on the magnitude of GC reactions or their capacity to produce memory B cell populations. However, the memory B cells generated in the absence of IL-21 exhibited reduced recall function upon challenge. Our data support that IL-21 prevents premature cellular dissolution within the GC and promotes stringency of selective pressures during B cell fate determination required to produce high-quality Plasmodium-specific memory B cells. These data are additionally consistent with a temporal requirement for IL-21 in fine-tuning humoral immune memory responses during experimental malaria.

Genome-resolved metagenomics reveals the effect of nutrient availability on bacterial genomic properties across 44 European freshwater lakes.

Understanding intricate microbial interactions in the environment is crucial. This is especially true for the relationships between nutrients and bacteria, as phosphorus, nitrogen and organic carbon availability are known to influence bacterial population dynamics. It has been suggested that low nutrient conditions prompt the evolutionary process of genome streamlining. This process helps conserve scarce nutrients and allows for proliferation. Genome streamlining is associated with genomic properties such as %GC content, genes encoding sigma factors, percent coding regions, gene redundancy, and functional shifts in processes like cell motility and ATP binding cassette transporters, among others. The current study aims to unveil the impact of nutrition on the genome size, %GC content, and functional properties of pelagic freshwater bacteria. We do this at finer taxonomic resolutions for many metagenomically characterized communities. Our study confirms the interplay of trophic level and genomic properties. It also highlights that different nutrient types, particularly phosphorus and nitrogen, impact these properties differently. We observed a covariation of functional traits with genome size. Larger genomes exhibit enriched pathways for motility, environmental interaction, and regulatory genes. ABC transporter genes reflect the availability of nutrients in the environment, with small genomes presumably relying more on metabolites from other organisms. We also discuss the distinct strategies different phyla adopt to adapt to oligotrophic environments. The findings contribute to our understanding of genomic adaptations within complex microbial communities.

Managing infectious challenges in the age of molecular-targeted therapies for adult hematological malignancies.

Over the last decade, the therapeutic landscape for hematological malignancies (HMs) has witnessed a remarkable surge in the development of novel biological and small-molecule-targeted immunomodulatory agents. These therapies have drastically improved survival, but some come at the cost of increased risk of bacterial, viral, and/or fungal infections and on-target off-tumor immunological side effects. To mitigate such risks, physicians must be well informed about infectious complications and necessary preventive measures, such as screening, vaccinations, and antimicrobial prophylaxis. Furthermore, physicians should be vigilant about the noninfectious side effects of these agents that can mimic infections and understand their potential drug-drug interactions with antimicrobials. Strengthening and harmonizing the current surveillance and reporting system for drug-associated infections in real-world settings is essential to better ascertain the potential infections associated with these agents. In this review, we aimed to summarize the infection risks associated with novel agents used for specific HMs and outline recommended strategies for monitoring and prophylaxis.

A guide to selecting upper thoracic versus lower thoracic uppermost instrumented vertebra in adult spinal deformity correction.

PURPOSE: Operative treatment of adult spinal deformity (ASD) has been shown to improve patient health-related quality of life (HRQOL). Selection of the uppermost instrumented vertebra (UIV) in either the upper thoracic (UT) or lower thoracic (LT) spine is a pivotal decision with effects on operative and postoperative outcomes. This review overviews the multifaceted decision-making process for UIV selection in ASD correction. METHODS: PubMed was queried for articles using the keywords "uppermost instrumented vertebra", "upper thoracic", "lower thoracic", and "adult spinal deformity". RESULTS: Optimization of UIV selection may lead to superior deformity correction, better patient-reported outcomes, and lower risk of proximal junctional kyphosis (PJK) and failure (PJF). Patient alignment characteristics, including preoperative thoracic kyphosis, coronal deformity, and the magnitude of sagittal correction influence surgical decision-making when selecting a UIV, while comorbidities such as poor body mass index, osteoporosis, and neuromuscular pathology should also be taken in to account. Additionally, surgeon experience and resources available to the hospital may also play a role in this decision. Currently, it is incompletely understood whether postoperative HRQOLs, functional and radiographic outcomes, and complications after surgery differ between selection of the UIV in either the UT or LT spine. CONCLUSION: The correct selection of the UIV in surgical planning is a challenging task, which requires attention to preoperative alignment, patient comorbidities, clinical characteristics, available resources, and surgeon-specific factors such as experience.

Comprehensive molecular profile of primary cutaneous epithelioid rhabdomyosarcoma: A tumor genomically and molecularly related to malignant melanoma.

The histogenesis of the rare primary cutaneous epithelioid rhabdomyosarcoma (PCERMS) remains unclear, with the morphological and immunophenotypic appearance of a rhabdomyosarcoma but a genomic profile consistent with sarcomatoid undifferentiated malignant melanoma (SUMM). Here, we provide comprehensive clinical, histopathological, and genomic analysis of a putative PCERMS presenting in an elderly patient. Histopathologic examination revealed an ulcerative tumefactive lesion with diffuse replacement of the dermis by sheets of malignant epithelioid cells with a rhabdoid appearance. By immunohistochemistry, the tumor cells were strongly and diffusely positive for desmin and myogenin. Comprehensive genomic analysis with a 542 gene DNA-based sequencing panel revealed likely biallelic NF1 inactivation (mutation and deletion), TERT promoter mutation, and a high tumor mutation burden (>100 mutations/mB) with features of a UV-mutational signature, which are all genomic features that can be seen in undifferentiated malignant melanoma. This case provides evidence of a close relationship at a molecular level between PCERMS and SUMM. Molecular genomic characterization of a larger cohort of PCERMS is warranted for further elucidation.

Artificial intelligence in bronchopulmonary dysplasia- current research and unexplored frontiers.

IMPACT: Provide an overview of bronchopulmonary dysplasia, its definitions, and their shortcomings. Explore the areas where machine learning may be used to further our understanding of bronchopulmonary dysplasia.

Electroconvulsive therapy for super refractory status epilepticus in pregnancy: case report and review of literature.

OBJECTIVE: We aim to describe use of electroconvulsive therapy (ECT) to treat super refractory status epilepticus (SRSE) in pregnancy and review the literature regarding utility and safety of ECT in refractory status epilepticus. BACKGROUND: Status epilepticus (SE) is a commonly encountered emergency in neuro-critical care world. Pharmacotherapy of status epilepticus in pregnancy is very challenging given the effect of the majority of antiepileptic drugs (AEDs) on fetal development. Although there has been growing evidence for use of ECT in status epilepticus, data about its utility in pregnancy is lacking. DESIGN/METHOD: A twenty-one year old Caucasian female with history of epilepsy presented at 8 weeks of gestation as status epilepticus (SE) after abrupt discontinuation of her AEDs. Treatment was initiated with standard regimen of benzodiazepine and levetiracetam, which was progressively expanded to include approximately 10 anti-epileptic drugs over the course of 30 days. The status epilepticus was super refractory to sedation. She underwent ECT on day 31 with remarkable improvement in electroencephalogram (EEG) pattern and resolution of status epilepticus following a single ECT session. We reviewed PubMed and collated case reports involving the use of ECT in status epilepticus with emphasis on differences in various confounding factors esp. etiology of status and age group. CONCLUSION: Our case is the first reported case of ECT for successful treatment of SRSE in pregnancy. While majority AEDs pose a significant maternal and fetal risk during pregnancy, ECT could be a potential frontline therapy for SE in pregnancy.

Groundwater arsenic contamination: impacts on human health and agriculture, ex situ treatment techniques and alleviation.

Groundwater is consumed by a large number of people as their primary source of drinking water globally. Among all the countries worldwide, nations in South Asia, particularly India and Bangladesh, have severe problem of groundwater arsenic (As) contamination so are on our primary focus in this study. The objective of this review study is to provide a viewpoint about the source of As, the effect of As on human health and agriculture, and available treatment technologies for the removal of As from water. The source of As can be either natural or anthropogenic and exposure mediums can either be air, drinking water, or food. As-polluted groundwater may lead to a reduction in crop yield and quality as As enters the food chain and disrupts it. Chronic As exposure through drinking water is highly associated with the disruption of many internal systems and organs in the human body including cardiovascular, respiratory, nervous, and endocrine systems, soft organs, and skin. We have critically reviewed a complete spectrum of the available ex situ technologies for As removal including oxidation, coagulation-flocculation, adsorption, ion exchange, and membrane process. Along with that, pros and cons of different techniques have also been scrutinized on the basis of past literatures reported. Among all the conventional techniques, coagulation is the most efficient technique, and considering the advanced and emerging techniques, electrocoagulation is the most prominent option to be adopted. At last, we have proposed some mitigation strategies to be followed with few long and short-term ideas which can be adopted to overcome this epidemic.

Krüppel-like factor 3 (KLF3) suppresses NF-κB-driven inflammation in mice.

Bacterial products such as lipopolysaccharides (or endotoxin) cause systemic inflammation, resulting in a substantial global health burden. The onset, progression, and resolution of the inflammatory response to endotoxin are usually tightly controlled to avoid chronic inflammation. Members of the NF-κB family of transcription factors are key drivers of inflammation that activate sets of genes in response to inflammatory signals. Such responses are typically short-lived and can be suppressed by proteins that act post-translationally, such as the SOCS (suppressor of cytokine signaling) family. Less is known about direct transcriptional regulation of these responses, however. Here, using a combination of in vitro approaches and in vivo animal models, we show that endotoxin treatment induced expression of the well-characterized transcriptional repressor Krüppel-like factor 3 (KLF3), which, in turn, directly repressed the expression of the NF-κB family member RELA/p65. We also observed that KLF3-deficient mice were hypersensitive to endotoxin and exhibited elevated levels of circulating Ly6C+ monocytes and macrophage-derived inflammatory cytokines. These findings reveal that KLF3 is a fundamental suppressor that operates as a feedback inhibitor of RELA/p65 and may be important in facilitating the resolution of inflammation.

Hit and Run Transcriptional Repressors Are Difficult to Catch in the Act.

Transcriptional silencing may not necessarily depend on the continuous residence of a sequence-specific repressor at a control element and may act via a "hit and run" mechanism. Due to limitations in assays that detect transcription factor (TF) binding, such as chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq), this phenomenon may be challenging to detect and therefore its prevalence may be underappreciated. To explore this possibility, erythroid gene promoters that are regulated directly by GATA1 in an inducible system are analyzed. It is found that many regulated genes are bound immediately after induction of GATA1 but the residency of GATA1 decreases over time, particularly at repressed genes. Furthermore, it is shown that the repressive mark H3K27me3 is seldom associated with bound repressors, whereas, in contrast, the active (H3K4me3) histone mark is overwhelmingly associated with TF binding. It is hypothesized that during cellular differentiation and development, certain genes are silenced by repressive TFs that subsequently vacate the region. Catching such repressor TFs in the act of silencing via assays such as ChIP-seq is thus a temporally challenging prospect. The use of inducible systems, epitope tags, and alternative techniques may provide opportunities for detecting elusive "hit and run" transcriptional silencing. Also see the video abstract here https://youtu.be/vgrsoP_HF3g.

Shotgun metagenome guided exploration of anthropogenically driven resistomic hotspots within Lonar soda lake of India.

Anthropogenic activities mediated antibiotic resistance genes (ARGs) in the pristine aquatic bodies (lakes) is raising concern worldwide. Long read shotgun sequencing was used to assess taxonomic diversity, distribution of ARGs and metal resistance genes (MRGs) and mobile genetic elements (MGEs) in six sites within hypersaline Lonar soda lake (India) prone to various anthropogenic activities. Proteobacteria and Euryarchaeota were dominant phyla under domain Bacteria and Archaea respectively. Higher abundance of Bacteroidetes was pragmatic at sites 18LN5 and 18LN6. Functional analysis indicated 26 broad-spectrum ARGs types, not reported earlier in this ecosystem. Abundant ARG types identified were multidrug efflux, glycopepetide, bacitracin, tetracycline and aminogylcoside resistance. Sites 18LN1 and 18LN5 depicted 167 and 160 different ARGs subtypes respectively and rpoB2, bcrA, tetA(48), mupA, ompR, patA, vanR and multidrug ABC transporter genes were present in all samples. The rpoB2 gene was dominant in 18LN1, whereas bcrA gene in 18LN2-18LN6 sites. Around 24 MRGs types were detected with higher abundance of arsenic in 18LN1 and copper in 18LN2-18LN6, signifying metal contamination linked to MRGs. The bacterial taxa Pseudomonas, Thioalkalivibrio, Burkholderia, Clostridium, Paenibacillus, Bacillus and Streptomyces were significantly associated with ARGs. This study highlights the resistomic hotspots in the lake for deploying policies for conservation efforts.

Untapped bacterial diversity and metabolic potential within Unkeshwar hot springs, India.

Hot springs support diverse and interesting groups of microorganisms adapted to extreme conditions and gaining attention in biotechnological applications. However, due to limitations of cultivation methods, a majority of such extremophiles remain uncultivated and unexplored. The advent of multiple cultivation conditions and specialized culture media could possibly aid to access the unexplored microbial portion of hot springs. In the present study, different media and isolation strategies were applied to isolate hitherto unexplored bacterial taxa in the water samples collected from Unkeshwar hot springs, India. Molecular, phylogenetic and predictive functional characterization of the isolated bacterial population was done using 16S rRNA sequencing coupled with Tax4Fun tools. Furthermore, representative isolates were screened for important enzymes (cellulase, xylanase, amylase, and protease) and heavy metal tolerance (chromium, arsenic) properties. A total of 454 bacterial isolates obtained were mapped into 57 unique bacterial genera and 4 different bacterial phyla. Interestingly, 37 genera not previously isolated from Indian hot springs, were isolated for the first time in the present study. However, most of these genera (23 out of 37) were reported only in metagenomics studies from Indian and global hot springs. Furthermore, around 14 genera not previously cultivated and not detected in metagenomics studies of hot springs are documented here. The metabolic potential was ascertained by determining the abundance of specific genes using in silico based Tax4Fun tool, which identified around 315 metabolic pathways for metabolism of carbohydrates, synthesis of secondary metabolites and degradation of xenobiotic compounds. Bioprospection study revealed that 33 and 25 bacterial genera were positive for enzyme production and resistance to the heavy metals, respectively. The present study revealed the advantages of cultivation methods using a comprehensive multiple isolation approach for exploring untapped and unique bacterial diversity, and also utilities for various biotechnological and environmental applications.

Efficacy and Safety of Flexible Endoscopic Management of Zenker's Diverticulum.

Zenker's diverticulum (ZD), a pulsion diverticulum of hypopharynx is a rare but treatable cause of morbidity in geriatric population. Traditionally a surgical disease but due to its associated high morbidity, flexible endoscopy has become a lucrative option. We reviewed 997 patients from 23 original studies who underwent flexible endoscopic diverticulotomy (FED) of ZD. Composite technical and clinical success rate for the study cohort was 99.4% and 87.9%, respectively. Composite failure rate was 10.0% but close to half of them (45.3%) had success with repeat endoscopic intervention. Composite rate for symptom recurrence after long-term follow-up was 13.6% but more than half (61.8%) had success with repeat endoscopic intervention. Bleeding (6.6%) and perforation (5.3%) were 2 most common complications of FED. All bleeding events were successfully managed with observation or endoscopic therapy. Majority of perforation events (4.4%) were successfully managed with conservative care and only 0.9% required invasive management. No mortality was reported. Efficacy and safety of FED of ZD remained same irrespective of diverticulum size or prior surgical/endoscopic treatment. FED with diverticuloscope (FEDD) and FED with cap (FEDC) had comparable technical success rate (99.6% vs. 100.0%) but FEDD had higher clinical success rate compared with FEDC (86.8% vs. 75.4%). FEDD had twice the risk of symptom recurrence than FEDC (16.5% vs. 9.5%). FEDD had a comparable bleeding risk to FEDC (3.3% vs. 4.0%) but a much lower perforation rate (2.3% vs. 10.3%). Upper esophageal sphincterotomy and adequate length of septotomy are the cornerstones of FED. FED can be considered a safe and efficacious treatment modality for patients with ZD.

Endoscopic Ultrasound Guided Choledocho-Enterostomy by Using Lumen Apposing Metal Stent in Patients with Failed Endoscopic Retrograde Cholangiopancreatography: A Literature Review.

BACKGROUND: Endoscopic retrograde cholangiopancreatography (ERCP) has been the cornerstone in the management of malignant jaundice to provide symptomatic relief and/or to allow chemotherapy. Difficult biliary cannulation or the presence of concomitant duodenal obstruction may lead to its failure. METHOD: An extensive English literature search was done via PubMed and Google Scholar to identify 13 peer-reviewed original articles. RESULTS: Of 92 patients, 98.9% (91/92) successfully underwent EUS-guided biliary drainage by placement of a lumen-apposing metal stent (LAMS) across extrahepatic biliary tree and small bowel. Although technically challenging, a similar technical success was noted for patients with limited dilation of extrahepatic biliary tree (less than 1 cm) and altered gastrointestinal anatomy (Roux-en-Y, Whipple, or Billroth II). Of 91 patients, 98.9% (90/91) had clinical success. Procedure complications included perforation (2/92; 2.2%) and bleeding (1/92; 1.1%). Stent complications included obstruction (7/91; 7.7%), and migration (1/91; 1.1%) with all responding to endoscopic debridement of food or tumor, placement of plastic pigtail stent through the LAMS, or replacement with a new stent. No procedure-related mortality was noted. CONCLUSION: In the hands of an expert, EUS-guided biliary drainage with the use of LAMS is an efficacious and safe option for patients with distal malignant biliary stricture, not amenable to ERCP.

Effect of Hyperosmolar Therapy on Outcome Following Spontaneous Intracerebral Hemorrhage: Ethnic/Racial Variations of Intracerebral Hemorrhage (ERICH) Study.

PURPOSE: We aimed to identify the effect of hyperosmolar therapy (mannitol and hypertonic saline) on outcomes after intracerebral hemorrhage (ICH) in the Ethnic/Racial Variations of Intracerebral Hemorrhage (ERICH) study. METHODS: Comparison of ICH cases treated with hyperosmolar therapy versus untreated cases was performed using a propensity score based on age, initial Glasgow Coma Scale, location of ICH (lobar, deep, brainstem, and cerebellar), log-transformed initial ICH volume, presence of intraventricular hemorrhage, and surgical interventions. ERICH subjects with a pre-ICH modified Rankin Scale (mRS) score of 3 or lower were included. Treated cases were matched 1:1 to untreated cases by the closest propensity score (difference ≤.15), gender, and race and ethnicity (non-Hispanic white, non-Hispanic black, or Hispanic). The McNemar and the Wilcoxon signed-rank tests were used to compare 3-month mRS outcomes between the 2 groups. Good outcome was defined as a 3-month mRS score of 3 or lower. RESULTS: As of December 31, 2013, the ERICH study enrolled 2279 cases, of which 304 hyperosmolar-treated cases were matched to 304 untreated cases. Treated cases had worse outcome at 3 months compared with untreated cases (McNemar, P = .0326), and the mean 3-month mRS score was lower in the untreated group (Wilcoxon, P = .0174). Post hoc analysis revealed more brain edema, herniation, and death at discharge for treated cases. CONCLUSIONS: Hyperosmolar therapy was not associated with better 3-month mRS outcomes for ICH cases in the ERICH study. This finding likely resulted from greater hyperosmolar therapy use in patients with edema and herniation rather than those agents leading to worse outcomes. Further studies should be performed to determine if hyperosmolar agents are effective in preventing poor outcomes.

What Is Actually Affected by the Scrambling of Objects When Localizing the Lateral Occipital Complex?

The lateral occipital complex (LOC), the cortical region critical for shape perception, is localized with fMRI by its greater BOLD activity when viewing intact objects compared with their scrambled versions (resembling texture). Despite hundreds of studies investigating LOC, what the LOC localizer accomplishes-beyond distinguishing shape from texture-has never been resolved. By independently scattering the intact parts of objects, the axis structure defining the relations between parts was no longer defined. This led to a diminished BOLD response, despite the increase in the number of independent entities (the parts) produced by the scattering, thus indicating that LOC specifies interpart relations, in addition to specifying the shape of the parts themselves. LOC's sensitivity to relations is not confined to those between parts but is also readily apparent between objects, rendering it-and not subsequent "place" areas-as the critical region for the representation of scenes. Moreover, that these effects are witnessed with novel as well as familiar intact objects and scenes suggests that the relations are computed on the fly, rather than being retrieved from memory.

Physician and Patient Preferences for Nonvalvular Atrial Fibrillation Therapies.

OBJECTIVES: The objective of this study was to compare patient and physician preferences for different antithrombotic therapies used to treat nonvalvular atrial fibrillation (NVAF). METHODS: Patients diagnosed with NVAF and physicians treating such patients completed 12 discrete choice questions comparing NVAF therapies that varied across five attributes: stroke risk, major bleeding risk, convenience (no regular blood testing/dietary restrictions), dosing frequency, and patients' out-of-pocket cost. We used a logistic regression to estimate the willingness-to-pay (WTP) value for each attribute. RESULTS: The 200 physicians surveyed were willing to trade off $38 (95% confidence interval [CI] $22 to $54] in monthly out-of-pocket cost for a 1% (absolute) decrease in stroke risk, $14 (95% CI $8 to $21) for a 1% decrease in major bleeding risk, and $34 (95% CI $9 to $60) for more convenience. The WTP value among 201 patients surveyed was $30 (95% CI $18 to $42) for reduced stroke risk, $16 (95% CI $9 to $24) for reduced bleeding risk, and -$52 (95% CI -$96 to -6) for convenience. The WTP value for convenience among patients using warfarin was $9 (95% CI $1 to $18) for more convenience, whereas patients not currently on warfarin had a WTP value of -$90 (95% CI -$290 to -$79). Both physicians' and patients' WTP value for once-daily dosing was not significantly different from zero. On the basis of survey results, 85.0% of the physicians preferred novel oral anticoagulants (NOACs) to warfarin. NOACs (73.0%) were preferred among patients using warfarin, but warfarin (78.2%) was preferred among patients not currently using warfarin. Among NOACs, both patients and physicians preferred apixaban. CONCLUSIONS: Both physicians and patients currently using warfarin preferred NOACs to warfarin. Patients not currently using warfarin preferred warfarin over NOACs because of an apparent preference for regular blood testing/dietary restrictions.

The Lateral Occipital Complex shows no net response to object familiarity.

In 1995, Malach et al. discovered an area whose fMRI BOLD response was greater when viewing intact, familiar objects than when viewing their scrambled versions (resembling texture). Since then hundreds of studies have explored this late visual region termed the Lateral Occipital Complex (LOC), which is now known to be critical for shape perception (James, Culham, Humphrey, Milner, & Goodale, 2003). Malach et al. (1995) discounted a role of familiarity by showing that "abstract" Henry Moore sculptures, unfamiliar to the subjects, also activated this region. This characterization of LOC as a region that responds to shape independently of familiarity has been accepted but never tested with control of the same low-level features. We assessed LOC's response to objects that had identical parts in two different arrangements, one familiar and the other novel. Malach was correct: There is no net effect of familiarity in LOC. However, a multivoxel correlation analysis showed that LOC does distinguish familiar from novel objects.

A Quantitative Fluorescence-Based Assay for Assessing LIM Domain-Peptide Interactions.

We have developed Förster resonance energy transfer (FRET)-based experiments for measuring the binding affinity, off-rates, and inferred on-rates for interactions between a family of transcriptional regulators and their intrinsically disordered binding partners. It was difficult to evaluate these interactions previously, as the transcriptional regulators are obligate binding proteins that aggregate in the absence of a binding partner. The assays rely on fusion constructs where binding domains are linked by a flexible tether containing a specific protease site, with fluorescent proteins at either end that display FRET when the complex is formed. Loss of FRET is monitored after cutting the tether followed by dilution or competition with a non-fluorescent peptide. These methods allowed a wide range of binding affinities (10-9 -10-5 m) to be determined. Our data indicate that interactions of closely related proteins can have surprisingly different binding properties.