RESUMEN

AIM: The aim of this study is to investigate the utility of interleukin-6 (IL-6) in the early diagnosis of serious bacterial infections (SBI) in febrile infants and to compare it with C-reactive protein (CRP). METHODS: Retrospective study conducted in the paediatric emergency department in Gothenburg, Sweden, on previously healthy, full-term infants aged ≤60 days with fever without a source (FWS) from 2014 to 2017. RESULTS: We included 536 infants with FWS, of whom IL-6 was analysed in 364 (68%) and CRP was analysed in 494 (92%). Approximately 70% of the infants presented with a fever duration of less than 12 h. The prevalence of SBIs was 14.8% (95% CI,11.3-18.9) in the IL-6 group and 17.8% (95% CI,14.5-21.5) in the CRP group. The sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) of IL-6 ≥50 ng/L were 93%, 66%, 98% and 33%, respectively. For CRP ≥20 mg/L, the sensitivity, specificity, NPV, and PPV were 76%, 89%, 95%, and 55%, respectively. Logistic regression analysis showed that CRP was significantly associated with SBI (p < 0.0001) in the entire population, whereas IL-6 was not. CONCLUSION: Interleukin-6 showed high sensitivity and NPV, which might assist in identifying SBIs early in febrile infants. However, IL-6 was not shown to be superior to CRP and further studies are needed to investigate whether IL-6 should be incorporated in clinical management.

RESUMEN

Biofilm-related biomaterial infections are notoriously challenging to treat and can lead to chronic infection and persisting inflammation. To date, a large body of research can be reviewed for coatings which potentially prevent bacterial infection while promoting implant integration. Yet only a very small number has been translated from bench to bedside. This study provides an in-depth analysis of the stability, antibacterial mechanism, and biocompatibility of medical grade polycaprolactone (mPCL), coated with human serum albumin (HSA), the most abundant protein in blood plasma, and tannic acid (TA), a natural polyphenol with antibacterial properties. Molecular docking studies demonstrated that HSA and TA interact mainly through hydrogen-bonding, ionic and hydrophobic interactions, leading to smooth and regular assemblies. In vitro bacteria adhesion testing showed that coated scaffolds maintained their antimicrobial properties over 3 days by significantly reducing S. aureus colonization and biofilm formation. Notably, amplitude modulation-frequency modulation (AMFM) based viscoelasticity mapping and transmission electron microscopy (TEM) data suggested that HSA/TA-coatings cause morphological and mechanical changes on the outer cell membrane of S. aureus leading to membrane disruption and cell death while proving non-toxic to human primary cells. These results support this antibiotic-free approach as an effective and biocompatible strategy to prevent biofilm-related biomaterial infections.

RESUMEN

Urinary stone disease (USD) is a prevalent urological condition, ranking as one of the most common urinary tract disorders globally. Various risk factors influence the formation of kidney stones, and recent research indicates a rising prevalence of urolithiasis worldwide, particularly in developing countries. While the morbidity associated with urinary stones has decreased in recent years, long-term complications such as stone recurrence, kidney failure, and uremia continue to burden patients. Understanding the etiologies of urolithiasis, including the role of bacteria, is crucial as they can contribute to stone recurrence. The incidence of urinary tract infection (UTI) stones can be attributed to specific infectious risk factors, socio-demographic factors, and comorbid metabolic disorders. This review article explores the emerging evidence suggesting the involvement of bacteria in USD. It discusses the potential role of microorganisms in non-infection stones and highlights the association between UTIs and urolithiasis. Furthermore, it surveys the relationship between kidney stones and recurrent UTIs and the formation of bacterial biofilms in UTIs. Considering various risk factors, including biochemical stone analysis and the presence of bacteria, is essential for treating patients with infectious stones optimally. This review aims to provide an updated understanding of the association between bacteria and urinary stones in patients with urolithiasis, shedding light on the pathophysiology of urinary stone formation, urinary stone characteristics, and the urinary microbiome in urinary stones.

RESUMEN

PURPOSE: Patients with bacterial, fungal, and viral community-acquired pneumonia (CAP) were studied to determine their metabolic profiles. METHODS: Loop-mediated isothermal amplification technology and nucleic acid sequence-dependent amplification combined with microfluidic chip technology were applied to screen multiple pathogens from respiratory tract samples. Eighteen patients with single bacterial infection (B-CAP), fifteen with single virus infection (V-CAP), twenty with single fungal infection (F-CAP), and twenty controls were enrolled. UHPLC-MS/MS analysis of untargeted serum samples for metabolic profiles. Multiple linear regression and Spearman's rank correlation analysis were used to determine associations between metabolites and clinical parameters. The sensitivity and specificity of the screened metabolites were also examined, along with their area under the curve. RESULTS: The metabolic signatures of patients with CAP infected by bacteria, viruses, and fungi were markedly different from those of controls. The abundances of 45, 56, and 79 metabolites were significantly unbalanced. Among these differential metabolites, 11, 13, and 29 were unique to the B-CAP, V-CAP, and F-CAP groups, respectively. Bacterial infections were the only known causes of disturbances in the pentose and glucuronate and aldarate and ascorbate metabolism interconversions metabolic pathway. CONCLUSIONS: Serum metabolomic techniques based on UHPLC-MS/MS may identify differences between individuals with CAP who have been infected by various pathogens, and they can also build a metabolite signature for early detection of the origin of infection and prompt care.

Asunto(s)

RESUMEN

BACKGROUND: Root resorption consists of complex, multistep processes that involve cell signalling caused by inflammation and stromal cells, which promotes the secretion of receptor activator of nuclear factor κB ligand/ macrophage-colony stimulating factor (RANKL/M-CSF) resulting in a resorptive process. OBJECTIVE: The aim of this narrative review was to analyse the literature related to root resorption resulting from microbial infection and to comparing it with non-microbial infection. METHODS: An electronic literature search was performed using the PubMed database and applying keywords of articles published in English. Eligible papers were reviewed to reveal the descriptions of bone and root resorption processes. The abstracts were searched manually to identify articles about infection-stimulating bone and root resorption. RESULTS: Three main types of root resorption were identified, two associated with primary bacterial infection and one secondary to bacterial infection. These include external inflammatory resorption, internal inflammatory resorption and external cervical (invasive) resorption. DISCUSSION: The magnitude of cytokine involvement that promotes resorption and M-CSF/RANKL production depends on multiple factors, including pathogen virulence, site of infection and host genetic factors that activate the inflammation at the infection site. Two mechanisms activate the resorption mechanisms-the canonical and non-canonical pathways that can activate clastic cells independently of the RANKL/RANK canonical pathways. CONCLUSIONS: Two pathways of root resorption co-exist in the body. When resorption is caused by infection, chronic inflammation due to bacterial infection prolongs the secretions of pro-inflammatory cytokines that intensify root and bone resorption. The second pathway is bacterial independent of the non-infection root resorption that is part of the wound healing process, which is limited in time due to its innate ability.

RESUMEN

The KEAP1-NFE2L2 axis is essential for the cellular response against metabolic and oxidative stress. KEAP1 is an adaptor protein of CUL3 (cullin 3) ubiquitin ligase that controls the cellular levels of NFE2L2, a critical transcription factor of several cytoprotective genes. Oxidative stress, defective autophagy and pathogenic infections activate NFE2L2 signaling through phosphorylation of the autophagy receptor protein SQSTM1, which competes with NFE2L2 for binding to KEAP1. Here we show that phosphoribosyl-linked serine ubiquitination of SQSTM1 catalyzed by SidE effectors of Legionella pneumophila controls NFE2L2 signaling and cell metabolism upon Legionella infection. Serine ubiquitination of SQSTM1 sterically blocks its binding to KEAP1, resulting in NFE2L2 ubiquitination and degradation. This reduces NFE2L2-dependent antioxidant synthesis in the early phase of infection. Levels of serine ubiquitinated SQSTM1 diminish in the later stage of infection allowing the expression of NFE2L2-target genes; causing a differential regulation of the host metabolome and proteome in a NFE2L2-dependent manner.

RESUMEN

OBJECTIVE: To explore the risk factors for pulmonary bacterial infection (PBI) after lung transplantation (LTX), and to evaluate the impact of PBI on the short-term postoperative mortality. METHODS: We retrospectively analyzed data on 549 recipients who underwent LTX at the Affiliated Wuxi People's Hospital of Nanjing Medical University, China, between January 2018 and December 2021. The risk factors for PBI after LTX were explored by univariate analysis and multivariate logistic regression. Cox proportional hazards regression models were used to estimate the hazard ratios (HR) and 95% confidence intervals (CI) of one-, two-, and three-year mortality. Subgroup analysis was performed by the time of postoperative PBI (≤7 days or 8-30 day after surgery). RESULTS: The incidence of postoperative PBI in 549 recipients was 82.70% (454/549). Preoperative history of infections with multidrug-resistant bacteria (OR 12.34, 95% CI 1.69-1572.39), Acinetobacter baumannii infection in donor (OR 3.08, 95% CI 1.26-9.66), and longer cold ischemia time (OR 1.16, 95% CI 1.03-1.32) were risk factors for postoperative PBI. Postoperative PBI was associated with one-year (HR 1.80, 95% CI 1.09-2.96), two-year (HR 1.91, 95% CI 1.20-3.04), and three-year mortality (HR 2.03, 95% CI 1.29-3.19). Subgroup analysis showed that PBI within 7 days after surgery was associated with one-year (HR 1.86, 95% CI 1.12-3.08), two-year (HR 1.99, 95% CI 1.25-3.17), and three-year mortality (HR 2.13, 95% CI 1.35-3.36), while PBI at 8-30 days after surgery was not associated with short-term mortality (one-year: HR 1.36, 95% CI 0.69-2.69; two-year: HR 1.48, 95% CI 0.80-2.76; three-year: HR 1.51, 95% CI 0.82-2.77). CONCLUSIONS: Donor-recipient and surgical factors are risk factors for PBI after LTX. Active prevention and treatment of PBI within the first 7 days after surgery may improve short-term survival.

RESUMEN

The alarming rise in global antimicrobial resistance underscores the urgent need for effective antibacterial drugs. Drawing inspiration from the bacterial-entrapment mechanism of human defensin 6, we have fabricated biomimetic peptide nanonets composed of multiple functional fragments for bacterial eradication. These biomimetic peptide nanonets are designed to address antimicrobial resistance challenges through a dual-approach strategy. First, the resulting nanofibrous networks trap bacteria and subsequently kill them by loosening the membrane structure, dissipating proton motive force, and causing multiple metabolic perturbations. Second, these trapped bacterial clusters reactivate macrophages to scavenge bacteria through enhanced chemotaxis and phagocytosis via the PI3K-AKT signaling pathway and ECM-receptor interaction. In vivo results have proven that treatment with biomimetic peptide nanonets can alleviate systemic bacterial infections without causing noticeable systemic toxicity. As anticipated, the proposed strategy can address stubborn infections by entrapping bacteria and awakening antibacterial immune responses. This approach might serve as a guide for the design of bioinspired materials for future clinical applications.

Asunto(s)

RESUMEN

Circular RNA (circRNA) is a type of single-stranded RNA that forms a covalently closed continuous loop, unlike linear RNA. The expression of circRNAs in mammals is often conserved across species and shows tissue and cell specificity. Some circRNA serve as gene regulators. However, the biological function of most circRNAs is unclear. CircRNA does not have 5' or 3' ends. The unique structure of circRNAs provides them with a much longer half-life and more resistance to RNase R than linear RNAs. Inflammatory lung responses occur in the pathogenesis and recovery of many lung diseases. Macrophages form the first line of host defense/innate immune responses and initiate/mediate lung inflammation. For example, in bacterial pneumonia, upon pro-inflammatory activation, they release early response cytokines/chemokines that recruit neutrophils, macrophages, and lymphocytes to sites of infection and clear pathogens. The functional effects and mechanisms by which circRNAs exert physiological or pathological roles in macrophage activation and lung inflammation remain poorly understood. In this article, we will review the current understanding and progress of circRNA biogenesis, regulation, secretion, and degradation. Furthermore, we will review the current reports on the role of circRNAs in macrophage activation and polarization, as well as in the process of inflammatory lung responses.

Asunto(s)

RESUMEN

Gasdermins (GSDMs) are proteins cleaved by caspase (CASP) to trigger pyroptosis. In teleosts, pyroptosis is mediated by gasdermin E (GSDME). The Pufferfish, Takifugu rubripes, possesses two GSDME orthologs: named TrGSDMEa and TrGSDMEb. TrGSDMEa is cleaved by CASP3/7 to liberate the N-terminal (NT) domain that can trigger pyroptosis in mammalian cells. However, the biological function of TrGSDMEa in pufferfish is unknown, and TrGSDMEb is poorly studied. We found that TrGSDMEb was cleaved by CASP1/3/6/7/8, but the resulting NT domain, despite its similarity to TrGSDMEa-NT domain in sequence and structure, failed to induce pyroptosis. TrGSDMEa and TrGSDMEb exhibited similar expression patterns in pufferfish under normal physiological conditions but were up- and downregulated, respectively, in expression during Vibrio harveyi and Edwardsiella tarda infection. Bacterial infection induced the activation of TrGSDMEa and CASP3/7 in pufferfish cells, resulting in pyroptosis accompanied with IL-1ß production and maturation. Inhibition of TrGSDMEa-mediated pyroptosis via TrCASP3/7 reduced the death of pufferfish cells and augmented bacterial dissemination in fish tissues. Structure-oriented mutagenesis identified 16 conserved residues in teleost GSDMEa that were required for the pore formation or auto-inhibition of GSDMEa. This study illustrates the role of GSDMEa-mediated pyroptosis in teleost defense against bacterial pathogens and provides new insights into the structure-based function of vertebrate GSDME. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-024-00237-x.

RESUMEN

Perioperative antibiotic prophylaxis (PAP) in lung transplant recipients (LuTRs) has high heterogeneity between centers. Our aim was to investigate retrospectively the approach to PAP in our center over a 20-year period (2002-2023), and its impact on early post-operative infections (EPOIs) after lung transplantation (LuT). Primary endpoint was diagnosis of EPOI, defined as any bacterial infection including donor-derived events diagnosed within 30 days from LuT. Main exposure variables were type of PAP (combination vs. monotherapy) and PAP duration. We enrolled 111 LuTRs. PAP consisted of single-agent or combination regimens in 26 (25.2%) and 85 (74.8%) LuTR. Median PAP duration was 10 days (IQR 6-13) days. Piperacillin/tazobactam was the most common agent used either as monotherapy (n = 21, 80.7%) or as combination with levofloxacin (n = 79, 92.9%). EPOIs were diagnosed in 30 (27%) patients. At multivariable analysis no advantages were found for combination regimens compared to single-agent PAP in preventing EPOI (OR: 1.57, 95% CI: 0.488-5.068, p:0.448). The impact of PAP duration on EPOIs development was investigated including duration of PAP ≤6 days as main exposure variables, without finding a significantly impact (OR:2.165, 95% CI: 0.596-7.863, p: 0.240). Our results suggest no advantages for combination regimens PAP in preventing EPOI in LuTR.

Asunto(s)

RESUMEN

Photocatalytic molecules are considered to be one of the most promising substitutions of antibiotics against multidrug-resistant bacterial infections. However, the strong excitonic effect greatly restricts their efficiency in antibacterial performance. Inspired by the interfacial dipole effect, a Ti3C2 MXene modified photocatalytic molecule (MTTTPyB) is designed and synthesized to enhance the yield of photogenerated carriers under light irradiation. The alignment of the energy level between Ti3C2 and MTTTPyB results in the formation of an interfacial dipole, which can provide an impetus for the separation of carriers. Under the role of a dipole electric field, these photogenerated electrons can rapidly migrate to the side of Ti3C2 for improving the separation efficiency of photogenerated electrons and holes. Thus, more electrons can be utilized to produce reactive oxygen species (ROS) under light irradiation. As a result, over 97.04% killing efficiency can be reached for Staphylococcus aureus (S. aureus) when the concentration of MTTTPyB/Ti3C2 was 50 ppm under 660 nm irradiation for 15 min. A microneedle (MN) patch made from MTTTPyB/Ti3C2 was used to treat the subcutaneous bacterial infection. This design of an organic-inorganic interface provides an effective method to minimize the excitonic effect of molecules, further expanding the platform of inorganic/organic hybrid materials for efficient phototherapy.

RESUMEN

Alzheimer's disease (AD) is one of the leading causes of dementia and is characterized by memory loss, mental and behavioral abnormalities, and impaired ability to perform daily activities. Even as a global disease that threatens human health, effective treatments to slow the progression of AD have not been found, despite intensive research and significant investment. In recent years, the role of infections in the etiology of AD has sparked intense debate. Pathogens invade the central nervous system through a damaged blood-brain barrier or nerve trunk and disrupt the neuronal structure and function as well as homeostasis of the brain microenvironment through a series of molecular biological events. In this review, we summarize the various pathogens involved in AD pathology, discuss potential interactions between pathogens and AD, and provide an overview of the promising future of anti-pathogenic therapies for AD.

RESUMEN

Eosinophils are a highly abundant cell type in the gastrointestinal tract during homeostatic conditions, where they have recently been reported to take on an activated phenotype following colonization by the bacterial microbiota. To date, there have been few studies investigating whether eosinophils respond to infection with enteric bacterial pathogens and/or investigating the requirements for eosinophils for effective bacterial pathogen control. In this study, we investigated the response of eosinophils to an acute enteric infection of mice with the bacterial pathogen Salmonella enterica serovar Typhimurium. We also assessed whether eosinophil deficiency impacted Salmonella burdens in the intestinal tract or impacted the systemic dissemination of Salmonella following an oral infection of littermate wild-type BALB/cJ and eosinophil-deficient ΔdblGATA BALB/cJ mice. We found comparable Salmonella burdens in the intestinal tract of wild-type and eosinophil-deficient mice and no significant differences in the levels of Salmonella disseminating to systemic organs within 3 days of infection. Despite our evidence suggesting that eosinophils are not an essential cell type for controlling bacterial burdens in this acute infection setting, we found higher levels of eosinophils in gut-draining lymph nodes following infection, indicating that eosinophils do respond to Salmonella infection. Our data contribute to the growing evidence that eosinophils are responsive to bacterial stimuli, yet the influence of and requirements for eosinophils during bacterial infection appear to be highly context-dependent.

RESUMEN

BACKGROUND: Kidney transplant recipients are at higher risk of infections due to immunosuppression, especially in the perioperative period after receiving induction therapy. Administration of iron has been linked to bacterial infections. This study investigated if receipt of intravenous iron at the time of kidney transplant increased bacterial infections post-transplant. METHODS: This single-center, retrospective study compared patients who received intravenous iron at the time of kidney transplant to those who did not. Patients were followed for 12 weeks after transplant. The primary outcome was incidence of bacterial infections following transplant; hemoglobin and transfusion needs were also examined. RESULTS: A total of 416 patients who received intravenous iron were compared to 416 patients who did not. Bacterial infections were similar between groups (14.4% iron group vs. 15.9% non-iron group). Intravenous iron did not influence bacterial infections on univariable or multivariable analyses when other infection confounders were accounted for. Patients who did not receive intravenous iron required more packed red blood cell transfusions in the 3 months following transplantation, but this was driven by factors other than intravenous iron as demonstrated by a post-hoc analysis. CONCLUSIONS: Intravenous iron did not increase the risk of bacterial infections in the immediate post-kidney transplant setting. Bacterial infections after transplant were associated with female sex, increasing age at transplant, receipt of transfusions, and increased duration of urinary catheters.

RESUMEN

Methicillin-resistant Staphylococcus aureus (MRSA) infections pose great challenges to skin wound care due to the severe drug resistance developed in the clinic. There is an urgent need to exploit next-generation bactericidal therapeutics that are both antibiotic-free and multifunctional for enhanced wound healing. Herein, we designed a Ca2+-crosslinked alginate hydrogel (EcNSIN@Alg) containing two naturally derived bioactive components, probiotics Escherichia coli Nissle1917 (EcN) and Squid ink nanoparticles (SIN), to treat MRSA-infected wounds. The injectable composite hydrogel displayed excellent biocompatibility, photothermal antibacterial activity, and reactive oxygen species (ROS) scavenging property. Importantly, the probiotic EcN can enhance the photothermal SIN to promote immune regulatory activities, shifting pro-inflammatory macrophages (M1) to anti-inflammatory macrophages (M2). In an MRSA-infected abscess model, EcNSIN@Alg can reduce the expression level of wound inflammatory factors and ROS, increase the number of anti-inflammatory macrophages, accelerate collagen deposition and promote wound healing. This work offers a new perspective on developing safe, antibiotic-free, multifunctional bactericides using fully bioderived materials, with potential applications in clinical practice.

RESUMEN

The present work focuses on the fabrication of Polyvinyl alcohol-chitosan-loaded oleanolic acid-nanofibers (PVA-CS-OLA-NFs) for bacterial infection. The prepared PVA-CS-OLA-NFs were characterized for contact angle, SEM, AFM, XRD, FTIR, and TGA. The solid-state characterization and in vitro performance evaluation of nanofibers reveal consistent interconnection and diameters ranging from 102 ±â€¯9.5 to 386 ±â€¯11.6 nm. The nanofibers have a flat surface topography and exhibit efficient drug entrapment. Moreover, the in vitro release profile of PVA-CS-OLA-NFs was found to be 51.82 ±â€¯1.49 % at 24 h. Furthermore, the hemocompatibility study showed that the developed PVA-CS-OLA-NFs are non-hemolytic to human blood. The PVA-CS-OLA-NFs demonstrate remarkable antibacterial capabilities, as evidenced by their MBC and MIC values, which range from 128 and 32 µg/mL, against the strains of S. aureus. The in-vivo fluorescence optical imaging showed the sustained PVA-CS-OLA-NFs release at the wound site infected with S. aureus for a longer duration of time. Moreover, the PVA-CS-OLA-NFs showed superior wound healing performance against S. aureus infected wounds compared to the marketed formulation. Further, the laser Doppler imaging system improved oxygen saturation, blood supply, and wound healing by providing real-time blood flow and oxygen saturation information.

RESUMEN

OBJECTIVE: This study aimed to assess the diagnostic accuracy of a novel marker, the combined lactate-to-glucose ratio (CLGR), in identifying cerebrospinal fluid (CSF) bacterial infection (CBI) in neurosurgical patients. Additionally, it seeks to establish cut-off values for CLGR and evaluate the reliability of measurement using blood gas analyzer (BGA). METHODS: CSF samples were collected from two neurosurgical centers in Kuala Lumpur, Malaysia, between January 2022 and October 2023. Conventional markers and CLGR were quantified using standard laboratory methods, with BGA utilized for measurement when feasible. Samples were categorized into confirmed CBI-positive (CBI+) and CBI-negative (CBI-) groups. Marker performance was compared, and receiver operating characteristic analysis conducted. Pearson's correlation assessed the agreement between BGA and laboratory measurements. RESULTS: Among the 130 CSF samples, 11 were CBI+. Both cLac and CLGR were significantly elevated in the CBI+ group (p<0.001). The area under the curve (AUC) for cLac and CLGR was 0.990 and 0.994, respectively. Using a cut-off of 6.0mmol/L, cLac demonstrated sensitivity of 100%, specificity of 93.3%, positive predictive value (PPV) of 57.9%, negative predictive value (NPV) of 100%, and diagnostic accuracy of 93.9%. CLGR ≥20.0 showed even higher accuracy: 100.0% sensitivity, 98.6% specificity, 84.6% PPV, 100% NPV, and overall accuracy of 98.5%. Both markers maintained excellent performance in blood-stained CSF. BGA measurements correlated well with laboratory results (r=0.980 & 0.999, respectively, p<0.001). CONCLUSIONS: CLac levels ≥6.0mmol/L and CLGR ≥20.0 accurately identified CBI in neurosurgical patients, with CLGR exhibiting superior efficacy. The potential for instant BGA measurement suggests promise for point-of-care testing.

RESUMEN

Shigellosis is an enteric infection that transmits through the faecal-oral route, which can occur during sex between men who have sex with men (MSM). Between 2009 and 2014, an epidemic of sexually transmissible Shigella flexneri 3a occurred in England that subsequently declined. However, from 2019 to 2021, despite SARS-CoV-2 restrictions, S. flexneri 3a continued to re-emerge. We explored possible drivers of re-emergence by comparing host demography and pathogen genomics. Cases were primarily among 35-64 year old men in London. Genomic analyses of 502 bacterial isolates showed that the majority (58%) of re-emerging MSM strains were a clonal replacement of the original, with reduced antimicrobial resistance, conservation of plasmid col156_1, and two SNPs with 19 predicted effects. The absence of major changes in the pathogen or host demographics suggest that other factors may have driven the re-emergence of S. flexneri 3a and highlight the need for further work in the area.