NKX3.1 Expression in Non-Prostatic Tumors and Characterizing its Expression in Esophageal/Gastroesophageal Adenocarcinoma.

The NKX3.1 immunohistochemical stain is widely recognized as a highly sensitive and specific marker for prostate adenocarcinoma. Nevertheless, its expression has been documented in various nonprostatic tissues and malignancies. This review aims to provide an overview of NKX3.1 expression in diverse tumor types, with a specific focus on its aberrant expression in esophageal/gastroesophageal adenocarcinoma (E/GE-ADC). In our investigation, we explored the expression of NKX3.1 in a series of E/GE-ADC to shed light on its prevalence in this tumor category. A total of 50 samples, comprising primary and metastatic E/GE-ADC specimens from 34 patients, were subjected to immunohistochemical analysis. Stained sections were scored based on the intensity and distribution-categorized as negative, weak, moderate, or strong in either a focal or diffuse pattern. Strong staining corresponds to the intensity observed in normal prostate controls, while focal and diffuse staining denote <50% and ≥50% of tumor nuclei staining positive, respectively. Our semiquantitative scoring revealed that 6 (12%) of the primary and metastatic E/GE-ADC specimens exhibited variable positivity for NKX3.1. This finding suggests that E/GE-ADC can sporadically stain positive for NKX3.1, introducing potential challenges in definitively determining the primary site of origin in certain clinical scenarios. Along with a literature review of NKX3.1 expression in other tumor types, our study provides additional important information about the extent to which this immunostain can be seen in E/GE-ADCs, which, to our knowledge, has not been reported.

Children with uncontrolled asthma and significant reversibility might show hypoxaemia.

Asthmatic children free of exacerbation with airway obstruction may have low partial pressure of oxygen (PaO2) which can be a marker for future risk, but PaO2 is scarcely measured during pulmonary function testing. We prospectively included asthmatic children with airway obstruction referred for pulmonary function testing, including blood gas analysis (n = 51). Hypoxaemia, defined as a value lower than - 2 z-score, was present in 15 (29%) children, and 37 (72%) children had a significant reversibility after bronchodilator administration. The multivariable model showed a positive influence of baseline forced expiratory volume in 1 s (FEV1) on PaO2 (ß coefficient 0.69, [95% CI: 0.07; 1.30]; P = 0.03), whereas uncontrolled asthma and FEV1 reversibility negatively influenced it (ß coefficient - 1.59 [95% CI: - 2.74; - 0.44]; P = 0.01; and - 0.07 [95% CI: - 0.13; - 0.02]; P = 0.01, respectively). As a consequence, children with uncontrolled symptoms of asthma and FEV1 reversibility ≥ 12% were significantly more at risk of having hypoxaemia compared to children with well/partly controlled asthma or no significant reversibility of FEV1.Conclusion: Among obstructive asthmatic children without current exacerbation, hypoxaemia is more likely to be seen in children with uncontrolled asthma and a significant post-bronchodilator FEV1 reversibility, in favour of different pathophysiology and treatment requirements of their airway obstruction.What is Known:• Recommendations are to treat asthmatic children in order to control respiratory symptom and maintain normal pulmonary function.• Asthmatic children free of exacerbation may have different pathophysiology for airway obstruction (central, peripheral, inflammatory, spasticity, remodelling) and should be treated according the pathophysiology of their airway disease.What is New:• In obstructive asthmatic children free of current exacerbation, the presence of hypoxaemia (ventilation-perfusion mismatch) is influenced by asthma control and post-bronchodilator reversibility, independently of the level of baseline airway obstruction.• The presence of hypoxaemia in obstructive asthmatic children free of current exacerbation can be highly suspected by the composite index "uncontrolled asthma + FEV1reversibility ≥ 12%" which may guide treatment.

Dermatofibrosarcoma Protuberans in a Patient With Cowden Syndrome: Revisiting the PTEN and PDGF Pathways.

PTEN hamartoma tumor syndrome, of which Cowden syndrome (CS) is the most recognized variant, is characterized by multiple benign and malignant tumors of ectodermal, mesodermal, and endodermal origins, secondary to germline mutation in the phosphatase and tensin homolog (PTEN) gene. Dermatofibrosarcoma protuberans (DFSP) is a locally aggressive malignant fibroblastic/myofibroblastic tumor of the skin, characterized by the t(17:22)(q22:q13) translocation resulting in fusion of the COL1A1 and PDGFB genes. An association between CS and DFSP has not been reported in the literature to date. The authors have encountered a male patient with CS and a history of DFSP that developed adjacent to a sclerotic fibroma on the parietal scalp, both excised at age 7. He presented at age 21 with an enlarging pink nodule at the same site on the parietal scalp. Excision revealed a dermal and subcutaneous storiform spindle cell proliferation with fat entrapment and positive staining for CD34, consistent with DFSP. Fluorescence in situ hybridization confirmed PDGFB gene rearrangement. PTEN expression in the patient's recurrent DFSP was nearly absent when compared with that of sporadic DFSP. To our knowledge, this is the first report of DFSP in a patient with CS. Although the association is likely to be coincidental, the authors revisited the PTEN and the PDGF pathways to speculate any possible interplay of the 2 conditions on a molecular level.

Expression of the p40 isoform of p63 has high specificity for cutaneous sarcomatoid squamous cell carcinoma.

Cutaneous spindle cell malignancies such as sarcomatoid squamous cell carcinoma (SCC), leiomyosarcoma, desmoplastic melanoma (DM) and atypical fibroxanthoma (AFX) may be morphologically indistinguishable, yet accurate diagnosis is important for appropriate clinical management. The distinction among these entities relies on immunohistochemical evaluation for epidermal, muscle or melanocytic differentiation. Epidermal differentiation markers include cytokeratins and p63. p63 is expressed as two distinct isoforms, ΔNp63 (p40) and TAp63. p40 positivity is highly specific for pulmonary SCC and head and neck sarcomatoid SCC. We examined the utility of p40 vs. p63 immunostaining in the differentiation of a variety of cutaneous spindle cell malignancies, including sarcomatoid SCC (n = 27), AFX (n = 34) and DM (n = 10). p40 was less sensitive than p63 for detecting sarcomatoid SCC (56% and 81%, respectively). p63 and p40 were comparably specific for sarcomatoid SCC relative to AFX, with only rare weak staining of tumor cells for p63 and/or p40 in a minority of AFX cases, including one case with approximately 10% of cells staining weakly for p40. All cases of DM were negative for p40 and p63. Our results support continued use of p63 for diagnosis of cutaneous sarcomatoid SCC because of greater sensitivity relative to p40.

Development of a tomato xylem-mimicking microfluidic system to study Ralstonia pseudosolanacearum biofilm formation.

The bacterial wilt pathogen Ralstonia pseudosolanacearum (Rps) colonizes plant xylem vessels and blocks the flow of xylem sap by its biofilm (comprising of bacterial cells and extracellular material), resulting in devastating wilt disease across many economically important host plants including tomatoes. The technical challenges of imaging the xylem environment, along with the use of artificial cell culture plates and media in existing in vitro systems, limit the understanding of Rps biofilm formation and its infection dynamics. In this study, we designed and built a microfluidic system that mimicked the physical and chemical conditions of the tomato xylem vessels, and allowed us to dissect Rps responses to different xylem-like conditions. The system, incorporating functional surface coatings of carboxymethyl cellulose-dopamine, provided a bioactive environment that significantly enhanced Rps attachment and biofilm formation in the presence of tomato xylem sap. Using computational approaches, we confirmed that Rps experienced linear increasing drag forces in xylem-mimicking channels at higher flow rates. Consistently, attachment and biofilm assays conducted in our microfluidic system revealed that both seeding time and flow rates were critical for bacterial adhesion to surface and biofilm formation inside the channels. These findings provided insights into the Rps attachment and biofilm formation processes, contributing to a better understanding of plant-pathogen interactions during wilt disease development.

Levels and Associated Factors of Clients' Satisfaction Toward Child Immunization at Grassroot Health Care Centers in Ho Chi Minh City, Vietnam.

Purpose: Immunization is the most cost-effective health strategy, contributing significantly to public health interventions for all ages, particularly for children. However, caregivers' satisfaction with immunization systems affects their decisions on immunization for their children. This study evaluated the levels of clients' satisfaction toward child immunization and to identify its associated factors. Methods: A cross-sectional study was conducted at 40 commune health centers (CHCs) in 24 districts in Ho Chi Minh City, Vietnam among 1200 caregivers of children aged under 5 years. Clients who took their children to CHCs for immunization were recruited based on convenience sampling technique and were asked to complete a self-report questionnaire. Satisfaction was measured using the Satisfaction with Immunization Service Questionnaire (SWISQ). Ordinal logistic regression models were fitted to identify factors associated with satisfaction levels. Results: The majority of participants were female (85.5%) with a mean age of 33.3 (standard deviation = 9.0). Approximately 60% of participants reported a moderate (40.2%) or high (17.1%) level of satisfaction. Participants with older children and those who waited for a longer duration had a lower satisfaction level. In contrast, high satisfaction level was found to be positive associated with being reminded by healthcare workers and the condition of follow-up areas, vaccine storage and the immunization process met participant's need. Conclusion: The level of clients' satisfaction toward child immunization at grassroot healthcare centers in Ho Chi Minh City is relatively low, with 40.2% having moderate satisfaction and 17.1% having high satisfaction. Strategies to improve vaccination programs at CHCs are needed, focusing on clients' experiences at CHCs during vaccination sessions. Further studies are also needed to have an in-depth understanding of more factors affecting satisfaction in this population.

Care for the Carers: An Evaluation of Job Satisfaction of Community Healthcare Workers in Charge of Infectious Disease Prevention and Control in Vietnam.

PURPOSE: This study explored job satisfaction and associated factors among community healthcare workers (HCWs) during the COVID-19 pandemic. METHODS: A cross-sectional study was conducted among 319 HCWs in charge of infectious disease prevention and control activities at all commune healthcare centers in Ho Chi Minh City. Participants completed a self-administered questionnaire which included the 36-item Job Satisfaction Survey (JSS). RESULTS: Most participants were male (56.7%), and the mean age was 34.7 (SD=7.1) years. The overall job satisfaction was relatively low. Among 9 aspects measured, coworker was found to have the highest level of satisfaction (19.6±3.9), followed by supervision (19.3±4.1). In contrast, the lowest level of satisfaction was observed in operating condition (11.4±3.4) and contingent rewards (14.3±3.8). The total score of the JSS indicated that only half of HCWs were satisfied with their job in general. Older male HCWs who were married and those who had higher income reported a higher level of job satisfaction in several aspects measured. However, there was no association between job satisfaction and other HCW's characteristics, including specialty, occupation type, and working experience. CONCLUSION: Since this is the crucial workforce in the battle against infectious diseases, urgent interventions are needed to increase job satisfaction in this population.

Preexisting TP53-Variant Clonal Hematopoiesis and Risk of Secondary Myeloid Neoplasms in Patients With High-grade Ovarian Cancer Treated With Rucaparib.

IMPORTANCE: A total of 1% to 3% of patients treated with a poly(adenosine diphosphate-ribose) polymerase inhibitor for high-grade ovarian cancer (HGOC) develop therapy-related myeloid neoplasms (t-MNs), which are rare but often fatal conditions. Although the cause of these t-MNs is unknown, clonal hematopoiesis of indeterminate potential (CHIP) variants can increase the risk of primary myeloid malignant neoplasms and are more frequent among patients with solid tumors. OBJECTIVES: To examine whether preexisting CHIP variants are associated with the development of t-MNs after rucaparib treatment and how these CHIP variants are affected by treatment. DESIGN, SETTING, AND PARTICIPANTS: This retrospective genetic association study used peripheral blood cell (PBC) samples collected before rucaparib treatment from patients in the multicenter, single-arm ARIEL2 (Study of Rucaparib in Patients With Platinum-Sensitive, Relapsed, High-Grade Epithelial Ovarian, Fallopian Tube, or Primary Peritoneal Cancer) (n = 491; between October 30, 2013, and August 9, 2016) and the multicenter, placebo-controlled, double-blind ARIEL3 (Study of Rucaparib as Switch Maintenance Following Platinum-Based Chemotherapy in Patients With Platinum-Sensitive, High-Grade Serous or Endometrioid Epithelial Ovarian, Primary Peritoneal or Fallopian Tube Cancer) (n = 561; between April 7, 2014, and July 19, 2016), which tested rucaparib as HGOC therapy in the treatment and maintenance settings, respectively. The follow-up data cutoff date was September 1, 2019. Of 1052 patients in ARIEL2 and ARIEL3, PBC samples from 20 patients who developed t-MNs (cases) and 44 randomly selected patients who did not (controls) were analyzed for the presence of CHIP variants using targeted next-generation sequencing. Additional longitudinal analysis was performed on available ARIEL2 samples collected during treatment and at the end of treatment. MAIN OUTCOMES AND MEASURES: Enrichment analysis of preexisting variants in 10 predefined CHIP-associated genes in cases relative to controls; association with clinical correlates. RESULTS: Among 1052 patients (mean [SE] age, 61.7 [0.3] years) enrolled and dosed in ARIEL2 and ARIEL3, 22 (2.1%) developed t-MNs. The t-MNs were associated with longer overall exposure to prior platinum therapies (13.2 vs 9.0 months in ARIEL2, P = .04; 12.4 vs 9.6 months in ARIEL3, P = .003). The presence of homologous recombination repair gene variants in the tumor, either germline or somatic, was associated with increased prevalence of t-MNs (15 [4.1%] of 369 patients with HGOC associated with an HRR gene variant vs 7 [1.0%] of 683 patients with wild-type HGOC, P = .002). The prevalence of preexisting CHIP variants in TP53 but not other CHIP-associated genes at a variant allele frequency of 1% or greater was significantly higher in PBCs from cases vs controls (9 [45.0%] of 20 cases vs 6 [13.6%] of 44 controls, P = .009). TP53 CHIP was associated with longer prior exposure to platinum (mean 14.0 months of 15 TP53 CHIP cases vs 11.1 months of 49 non-TP53 CHIP cases; P = .02). Longitudinal analysis showed that preexisting TP53 CHIP variants expanded in patients who developed t-MNs. CONCLUSIONS AND RELEVANCE: The findings of this genetic association study suggest that preexisting TP53 CHIP variants may be associated with t-MNs after rucaparib treatment.

Molecular and clinical determinants of response and resistance to rucaparib for recurrent ovarian cancer treatment in ARIEL2 (Parts 1 and 2).

ARIEL2 (NCT01891344) is a single-arm, open-label phase 2 study of the PARP inhibitor (PARPi) rucaparib in relapsed high-grade ovarian carcinoma. In this post hoc exploratory biomarker analysis of pre- and post-platinum ARIEL2 samples, RAD51C and RAD51D mutations and high-level BRCA1 promoter methylation predict response to rucaparib, similar to BRCA1/BRCA2 mutations. BRCA1 methylation loss may be a major cross-resistance mechanism to platinum and PARPi. Genomic scars associated with homologous recombination deficiency are irreversible, persisting even as platinum resistance develops, and therefore are predictive of rucaparib response only in platinum-sensitive disease. The RAS, AKT, and cell cycle pathways may be additional modulators of PARPi sensitivity.

BRCA Reversion Mutations in Circulating Tumor DNA Predict Primary and Acquired Resistance to the PARP Inhibitor Rucaparib in High-Grade Ovarian Carcinoma.

A key resistance mechanism to platinum-based chemotherapies and PARP inhibitors in BRCA-mutant cancers is the acquisition of BRCA reversion mutations that restore protein function. To estimate the prevalence of BRCA reversion mutations in high-grade ovarian carcinoma (HGOC), we performed targeted next-generation sequencing of circulating cell-free DNA (cfDNA) extracted from pretreatment and postprogression plasma in patients with deleterious germline or somatic BRCA mutations treated with the PARP inhibitor rucaparib. BRCA reversion mutations were identified in pretreatment cfDNA from 18% (2/11) of platinum-refractory and 13% (5/38) of platinum-resistant cancers, compared with 2% (1/48) of platinum-sensitive cancers (P = 0.049). Patients without BRCA reversion mutations detected in pretreatment cfDNA had significantly longer rucaparib progression-free survival than those with reversion mutations (median, 9.0 vs. 1.8 months; HR, 0.12; P < 0.0001). To study acquired resistance, we sequenced 78 postprogression cfDNA, identifying eight additional patients with BRCA reversion mutations not found in pretreatment cfDNA. SIGNIFICANCE: BRCA reversion mutations are detected in cfDNA from platinum-resistant or platinum-refractory HGOC and are associated with decreased clinical benefit from rucaparib treatment. Sequencing of cfDNA can detect multiple BRCA reversion mutations, highlighting the ability to capture multiclonal heterogeneity.This article is highlighted in the In This Issue feature, p. 151.

Release and Constancy of an Antibiotic Resistance Gene in Seawater under Grazing Stress by Ciliates and Heterotrophic Nanoflagellates.

Extracellular DNA (exDNA) is released from bacterial cells through various processes. The antibiotic resistance genes (ARGs) coded on exDNA may be horizontally transferred among bacterial communities by natural transformation. We quantitated the released/leaked tetracycline resistance gene, tet(M) over time under grazing stress by ciliates and heterotrophic nanoflagellates (HNFs), and found that extracellular tet(M) (ex-tetM) increased with bacterial grazing. Separate microcosms containing tet(M)-possessing bacteria with ciliates or HNFs were prepared. The copy number of ex-tetM in seawater in the ciliate microcosm rapidly increased until 3 d after the incubation, whereas that in the HNF microcosm showed a slower increase until 20 d. The copy number of ex-tetM was stable in both cases throughout the incubation period, suggesting that extracellular ARGs are preserved in the environment, even in the presence of grazers. Additionally, ARGs in bacterial cells were constant in the presence of grazers. These results suggest that ARGs are not rapidly extinguished in a marine environment under grazing stress.

Biocompatibility Assessment of Detonation Nanodiamond in Non-Human Primates and Rats Using Histological, Hematologic, and Urine Analysis.

Detonation nanodiamonds (DNDs) have been widely explored for biomedical applications ranging from cancer therapy to magnetic resonance imaging due to several promising properties. These include faceted surfaces that mediate potent drug binding and water coordination that have resulted in marked enhancements to the efficacy and safety of drug delivery and imaging. In addition, scalable processing of DNDs yields uniform particles. Furthermore, a broad spectrum of biocompatibility studies has shown that DNDs appear to be well-tolerated. Prior to the clinical translation of DNDs for indications that are addressed via intravenous administration, comprehensive assessment of DND safety in both small and large animal preclinical models is needed. This article reports the results of a DND biocompatibility study in both non-human primates and rats. The rat study was performed as a multiple dose subacute investigation in two cohorts that lasted for 2 weeks and included histological, serum, and urine analysis. The non-human primate study was performed as a dual gender, multiple dose, and long-term investigation in both standard/clinically relevant and elevated dosing cohorts that lasted for 6 months and included comprehensive serum, urine, histological, and body weight analysis. The results from these studies indicate that NDs are well-tolerated at clinically relevant doses. Examination of dose-dependent changes in biomarker levels provides important guidance for the downstream in-human validation of DNDs for clinical drug delivery and imaging.

Unclassified renal cell carcinoma with tubulopapillary architecture, clear cell phenotype, and chromosome 8 monosomy: a new kid on the block.

Accurate subtyping of renal cell carcinomas (RCCs) has become clinically important for therapy and prognostication. RCC subtypes are defined by distinct morphologic and immunohistochemical profiles, and in some instances recurrent cytogenetic and molecular properties. However, some tumors exhibit overlapping morphologic and immunophenotypic features, frequent enough to pose diagnostic dilemmas. This report concerns six histologically unusual RCCs that showed tubulopapillary architecture, clear cell phenotype, and non-diagnostic immunohistochemical profiles. Further investigation of these tumors utilized a single nucleotide polymorphism (SNP) microarray platform (OncoScan®, Affymetrix) that employed molecular inversion probe (MIP) technology to investigate genome-wide chromosomal copy number changes and loss of heterozygosity in formalin-fixed paraffin-embedded sections. The six tumors were assayed in parallel with and in comparison to RCC with typical morphologic or immunohistochemical features for a specific subtype (clear cell, clear cell papillary, and microphthalmia transcription factor (MiT) family translocation RCC). Three of the unusual RCCs showed a molecular signature of clear cell RCC and one of papillary RCC. The remaining two showed monosomy of chromosome 8. Those two cases were tested via next-generation sequencing, and no pathogenic variants were detected, including those in the genes VHL, PBRM1, SETD2, KDM5C, or BAP1. The addition of molecular investigations such as reported here as applied to histologically and immunohistochemically unusual RCC may help to define additional subtypes and contribute to the development of targeted therapy for renal cancer.

Persistence of Multi-Drug Resistance Plasmids in Sterile Water under Very Low Concentrations of Tetracycline.

The persistence of the multi-drug resistance plasmids pAQU1 and IncFIB was examined in bacterial populations under very low selective pressure. We herein demonstrated that these plasmids stably remained not only in the original host, but also in a transconjugant, even after being in a non-culturable state. In seawater microcosms containing Photobacterium damselae 04Ya311 possessing pAQU1, no significant loss of pAQU1 was observed during a 30-d starvation period. The copy numbers of pAQU1 and IncFIB in E. coli were constant. The results of the present study suggest that these plasmids have the ability to remain among various bacteria under oligotrophic conditions with low antibiotic selection pressure.

Controversies and considerations in the diagnosis of primary cutaneous CD4⁺ small/medium T-cell lymphoma.

CONTEXT: Primary cutaneous CD4⁺ small/medium T-cell lymphoma is a provisional and controversial entity with a broad differential diagnosis. Despite being an uncommon lymphoma, it is a frequent diagnostic consideration in cutaneous biopsies with a dense lymphoid infiltrate because it shows overlapping features with reactive lymphoid hyperplasia (pseudolymphoma) and a variety of other primary cutaneous and systemic lymphomas. However, proper classification of this process is important for determining patient prognosis and treatment options. OBJECTIVE: To review the clinical, morphologic, immunophenotypic, and genetic features of primary cutaneous CD4⁺ small/medium T-cell lymphoma and contrast those features with entities in the differential diagnosis. DATA SOURCES: Applicable literature will be reviewed with emphasis on current controversies and distinguishing characteristics. CONCLUSIONS: Although many consider primary cutaneous CD4⁺ small/medium T-cell lymphoma to be indistinguishable from reactive lymphoid hyperplasia/pseudolymphoma, it can be differentiated from other primary cutaneous and systemic lymphomas. Patients with solitary lesions of primary cutaneous CD4⁺ small/medium T-cell lymphoma generally have an excellent prognosis. Nevertheless, a subset of patients who have been reported to meet criteria for this lymphoma have followed a more-aggressive course; however, those patients show some differing clinical, morphologic, and immunophenotypic features.

Case series of volar juvenile xanthogranuloma: clinical observation of a peripheral rim of hyperkeratosis.

Juvenile xanthogranuloma is a benign histiocytic tumor predominantly occurring in children as yellowish papules on the head and trunk. Presentations on the volar surfaces are rare and may cause diagnostic confusion with pyogenic granuloma, eccrine poroma and digital fibrokeratoma. We report two patients with unusual presentations of solitary juvenile xanthogranuloma on the palm or sole. Both had lesions lacking the classic yellowish color and demonstrating a well-defined, peripheral hyperkeratotic rim. Histopathological evaluation revealed prominent orthokeratosis corresponding to the rim. Additional histological features, including dermal histiocytes and Touton giant cells, were consistent with the diagnosis of juvenile xanthogranuloma. Given the unusual locations and colors of the lesions, we conclude that histopathological evaluation is central to diagnosing volar juvenile xanthogranuloma. We additionally suggest that juvenile xanthogranuloma should be included in the differential diagnoses of volar lesions displaying a peripheral hyperkeratotic rim.

Secretion of heterologous thermostable cellulases in Bacillus subtilis.

Bacillus subtilis is used industrially for the production of secreted enzymes. The most characteristic feature of the secreted enzymes is variation in the N-terminal signal peptides that is recognized by secretion machinery, which is one of the determinants of efficiency and must be customized in each case. Culturing cellulolytic B. subtilis to secrete heterologous cellulases combined with customized signal peptides would be beneficial for producing biocommodities from cellulosic biomass. Four Clostridium thermocellum genes, encoding endoglucanases (celA and celB) and exoglucanases (celK and celS) were cloned to construct random libraries of combinations with 173 different signal peptides originating from the B. subtilis genome. The libraries were successfully screened to identify the signal peptides most efficient in secretion of each of the four cellulases, which were theoretically unpredictable. The secreted cellulases were assayed on carboxymethyl cellulose, phosphoric acid swollen cellulose, and microcrystalline cellulose to determine the possible effects of the signal peptides on substrate specificity. The customized signal peptides for CelA, CelB, and CelS did not affect enzyme performance but those for CelK might influence its substrate specificity.