Evaluating the efficacy of percutaneous puncture biopsy guided by contrast-enhanced ultrasound for peripheral pulmonary lesions.

BACKGROUND: The incidence and mortality of lung cancer have increased annually. Accurate diagnosis can help improve therapeutic efficacy of interventions and prognosis. Percutaneous lung biopsy is a reliable method for the clinical diagnosis of lung cancer. Ultrasound-guided percutaneous lung biopsy technology has been widely promoted and applied in recent years. AIM: To investigate the diagnostic value of contrast-enhanced ultrasound (CEUS)-guided percutaneous biopsy in peripheral pulmonary lesions. METHODS: We retrospectively collected data on 237 patients with peripheral thoracic focal lesions who underwent puncture biopsy at Wuxi People's Hospital. The patients were randomly divided into two groups: The CEUS-guided before lesion puncture group (contrast group) and conventional ultrasound-guided group (control group). Analyze the diagnostic efficacy of the puncture biopsy, impact of tumor size, and number of puncture needles and complications were analyzed and compared between the two groups. RESULTS: Accurate pathological results were obtained for 92.83% (220/237) of peripheral lung lesions during the first biopsy, with an accuracy rate of 95.8% (113/118) in the contrast group and 89.9% (107/119) in the control group. The difference in the area under the curve (AUC) between the contrast and the control groups was not statistically significant (0.952 vs 0.902, respectively; P > 0.05). However, when the lesion diameter ≥ 5 cm, the diagnostic AUC of the contrast group was higher than that of the control group (0.952 vs 0.902, respectively; P < 0.05). In addition, the average number of puncture needles in the contrast group was lower than that in the control group (2.58 ± 0.53 vs 2.90 ± 0.56, respectively; P < 0.05). CONCLUSION: CEUS guidance can enhance the efficiency of puncture biopsy of peripheral pulmonary lesions, especially for lesions with a diameter ≥ 5 cm. Therefore, CEUS guidance has high clinical diagnostic value in puncture biopsy of peripheral focal lung lesions.

Single-nucleus transcriptome unveils the role of ferroptosis in ischemic stroke.

Multiple cell death pathways are involved in neuronal death in ischemic stroke (IS). However, the role of different cell death pathways in different cell types has not been elucidated. By analyzing three single-nucleus RNA sequencing (snRNA-seq) data of IS, we first found that a variety of programmed cell death (PCD) -related genes were significantly changed in different cell types. Based on machine learning and virtual gene knockout, we found that ferroptosis related genes, ferritin heavy chain 1 (Fth1) and ferritin light chain (Ftl1), play a key role in IS. Ftl1 and Fth1 can promote microglia activation, as well as the production of inflammatory factors and chemokines. Cell communication analysis showed that activated microglia could enhance chemotactic peripheral leukocyte infiltration, such as macrophages and neutrophils, through Spp1-Cd44 and App-Cd74 signaling, thereby aggravating brain tissue damage. Furthermore, real-time quantitative polymerase chain reaction (RT-qPCR) showed that P2ry12 and Mef2c were significantly decreased in oxygen-glucose deprivation (OGD) group, while Ftl1, Fth1, Apoe, Ctsb, Cd44 and Cd74 were significantly increased in OGD group. Collectively, our findings suggested targeted therapy against microglia Ftl1 and Fth1 might improve the state of microglia, reduce the infiltration of peripheral immune cells and tissue inflammation, and then improve the ischemic brain injury in mouse.

Complement factor I knockdown inhibits colon cancer development by affecting Wnt/ß-catenin/c-Myc signaling pathway and glycolysis.

BACKGROUND: Colon cancer (CC) occurrence and progression are considerably influenced by the tumor microenvironment. However, the exact underlying regulatory mechanisms remain unclear. AIM: To investigate immune infiltration-related differentially expressed genes (DEGs) in CC and specifically explored the role and potential molecular mechanisms of complement factor I (CFI). METHODS: Immune infiltration-associated DEGs were screened for CC using bioinformatics. Quantitative reverse transcription polymerase chain reaction was used to examine hub DEGs expression in the CC cell lines. Stable CFI-knockdown HT29 and HCT116 cell lines were constructed, and the diverse roles of CFI in vitro were assessed using CCK-8, 5-ethynyl-2'-deoxyuridine, wound healing, and transwell assays. Hematoxylin and eosin staining and immunohistochemistry staining were employed to evaluate the influence of CFI on the tumorigenesis of CC xenograft models constructed using BALB/c male nude mice. Key proteins associated with glycolysis and the Wnt pathway were measured using western blotting. RESULTS: Six key immune infiltration-related DEGs were screened, among which the expression of CFI, complement factor B, lymphoid enhancer binding factor 1, and SRY-related high-mobility-group box 4 was upregulated, whereas that of fatty acid-binding protein 1, and bone morphogenic protein-2 was downregulated. Furthermore, CFI could be used as a diagnostic biomarker for CC. Functionally, CFI silencing inhibited CC cell proliferation, migration, invasion, and tumor growth. Mechanistically, CFI knockdown downregulated the expression of key glycolysis-related proteins (glucose transporter type 1, hexokinase 2, lactate dehydrogenase A, and pyruvate kinase M2) and the Wnt pathway-related proteins (ß-catenin and c-Myc). Further investigation indicated that CFI knockdown inhibited glycolysis in CC by blocking the Wnt/ß-catenin/c-Myc pathway. CONCLUSION: The findings of the present study demonstrate that CFI plays a crucial role in CC development by influencing glycolysis and the Wnt/ß-catenin/c-Myc pathway, indicating that it could serve as a promising target for therapeutic intervention in CC.

Development of nanogap-rich hybrid gold nanostructures by use of two non-lithographic deposition techniques for a sensitive and reliable SERS biosensor.

Practical application of surface-enhanced Raman spectroscopy (SERS) has suffered from several limitations by heterogeneous distribution of hot-spots, such as high signal fluctuation and the resulting low reliability in detection. Herein, we develop a strategy of more sensitive and reliable SERS platform through designing spatially homogeneous gold nanoparticles (GNPs) on a uniform gold nanoisland (GNI) pattern. The proposed SERS substrate is successfully fabricated by combining two non-lithographic techniques of electron beam evaporation and convective self-assembly. These bottom-up methods allow a simple, cost-effective, and large-area fabrication. Compared to the SERS substrates obtained from two separate nanofabrication methods, Raman spectra measured by the samples with both GNPs and GNIs present a significant increase in the signal intensity as well as a notable improvement in signal fluctuation. The simulated near-field analyses demonstrate the formation of highly amplified plasmon modes within and at the gaps of the GNP-GNI interfaces. Moreover, the suggested SERS sensor is evaluated to detect the glucose concentration, exhibiting that the detection sensitivity is improved by more than 10 times compared to the sample with only GNI patterns and a fairly good spatial reproducibility of 7% is accomplished. It is believed that our suggestion could provide a potential for highly sensitive, low-cost, and reliable SERS biosensing platforms that include many advantages for healthcare devices. Supplementary Information: The online version contains supplementary material available at 10.1007/s13534-024-00381-4.

Tankyrase 2 promotes lung cancer cell malignancy.

BACKGROUND: Tankyrase 2 (TNKS2) is a potential candidate molecular target for the prognosis and treatment of non-small cell lung cancer (NSCLC), but its biological functions are unclear. AIM: To investigate the biological functions of TNKS2 in NSCLC. METHODS: Using a lentiviral vector, we generated H647 model cells with TNKS2 knockdown by RNA interference and A549 model cells with TNKS2 overexpression by transfection with a TNKS2 overexpressing plasmid. Increased and decreased expression levels of TNKS2 in the two cell lines were verified using real-time reverse transcriptase-polymerase chain reaction and Western blot analyses. Cell apoptosis, proliferation, and migration were determined using flow cytometry, carboxyfluorescein succinimidyl ester staining, and scratch assay, respectively. Immunofluorescence staining was conducted to examine TNKS2 and ß-catenin expression levels in the two transfected cell lines and the non-transfected cells. RESULTS: TNKS2 mRNA and protein expression was significantly higher in the highly malignant NCI-H647 cells, while it remained at a low level in the less malignant A549 cells. Lentivirus-mediated overexpression of TNKS2 in A549 cells resulted in a 3-fold increase in gene expression and a 1.7-fold increase in protein expression (P < 0.01). Conversely, shRNA interference targeting TNKS2 Led to an 8-fold decrease in gene expression and a 3-fold decrease in protein expression (P < 0.01) in NCI-H647 cells. Furthermore, the cell apoptosis rate was significantly reduced (50%) and cell migration rate was increased (35%) in the TNKS2 overexpression group than in the control group (P < 0.05). In contrast, shTNKS2 promoted apoptosis by more than one fold and reduced migration by 60% (P < 0.05). Immunofluorescence analysis revealed enhanced nuclear localization of ß-catenin fluorescence signal associated with high TNKS2 expression levels. Western blot analysis investigating TNKS2/ß-catenin-related proteins indicated consistent changes between TNKS2 and ß-catenin expression in lung cancer cells, whereas Axin displayed an opposite trend (P < 0.05). CONCLUSION: The obtained results revealed that TNKS2 may serve as an adverse prognostic factor and a potential therapeutic target in NSCLC.

Improvement in Performance and Stability of PbS QD/IGZO Phototransistors Through the Introduction of Ga2O3 Film for Broadband Sensor Applications.

The development of broadband photosensors has become crucial in various fields. Indium-gallium-zinc oxide (IGZO, In:Ga:Zn = 1:1:1) phototransistors with PbS quantum dots (QDs) have shown promising features for such sensors, such as reasonable mobility, low leakage current, good photosensitivity, and low-cost fabrication. However, the instability of PbS QD/IGZO phototransistors under an air atmosphere and prolonged storage remain serious concerns. In this article, two concepts to improve the reliability of PbS QD/IGZO phototransistors were implemented. P-type doping in the PbS QD layer through oxidation allows increasing the built-in potential between IGZO and PbS QDs, leading to enhancement in photoinduced electron-hole pair creation. Second, agglomeration and fusion of a PbS QDs layer were controlled via thermal annealing, which facilitated the transport of photocreated carriers. The p-type doping and interconnection of a PbS QD layer can be achieved by deposition and subsequent thermal annealing of gallium oxide (Ga2O3) on PbS QD/IGZO stacks. The resulting Ga2O3/PbS QD/IGZO phototransistors exhibited high-performance switching characteristics under dark conditions. Notably, they showed a remarkable photoresponsivity of 196.69 ± 4.05 A/W and a detectivity of (5.47 ± 1.4) × 1012 Jones even at a long-wavelength illumination of 1550 nm. While the unpassivated PbS/IGZO phototransistor suffered serious degradation in optical performance after 2 weeks of storage, the Ga2O3/PbS QD/IGZO phototransistor demonstrated enhanced stability, maintaining high performance for over 5 weeks. These findings suggest that Ga2O3/PbS QD/IGZO phototransistors offer a feasible approach for the fabrication of large-scale active matrix broadband photosensor arrays, potentially revolutionizing optical sensing in various cutting-edge applications.

New Monoterpenoid Glycosides from the Fruits of Hypericum patulum Thunb.

The whole Hypericum patulum Thunb. plant is utilized in traditional medicine for its properties of clearing heat, detoxifying, soothing meridians, relaxing the liver, and stopping bleeding. In folk medicine, it is frequently used to treat hepatitis, colds, tonsillitis, and bruises. Phytochemical investigation of a 30% ethanol extract of the fresh ripe fruits of H. patulum has resulted in the isolation of two new pinane-type monoterpenoid glycosides 1-2, named patulumside E-F, and three new chain-shaped monoterpenoid glycosides 3-5, named patulumside G-H, J. Their structures were determined using extensive spectroscopic techniques, such as HR-ESI-MS, 1D and 2D NMR spectroscopy, and electronic circular dichroism (ECD) calculation. The anti-inflammatory activities of these compounds were evaluated in the LPS-induced RAW264.7 cells. This research represents the inaugural comprehensive phytochemical study of H. patulum, paving the way for further exploration of monoterpenoid glycosides.

Identification of human host factors required for beta-defensin-2 expression in intestinal epithelial cells upon a bacterial challenge.

The human intestinal tract is colonized with microorganisms, which present a diverse array of immunological challenges. A number of antimicrobial mechanisms have evolved to cope with these challenges. A key defense mechanism is the expression of inducible antimicrobial peptides (AMPs), such as beta-defensins, which rapidly inactivate microorganisms. We currently have a limited knowledge of mechanisms regulating the inducible expression of AMP genes, especially factors from the host required in these regulatory mechanisms. To identify the host factors required for expression of the beta-defensin-2 gene (HBD2) in intestinal epithelial cells upon a bacterial challenge, we performed a RNAi screen using a siRNA library spanning the whole human genome. The screening was performed in duplicate to select the strongest 79 and 110 hit genes whose silencing promoted or inhibited HBD2 expression, respectively. A set of 57 hits selected among the two groups of genes was subjected to a counter-screening and a subset was subsequently validated for its impact onto HBD2 expression. Among the 57 confirmed hits, we brought out the TLR5-MYD88 signaling pathway, but above all new signaling proteins, epigenetic regulators and transcription factors so far unrevealed in the HBD2 regulatory circuits, like the GATA6 transcription factor involved in inflammatory bowel diseases. This study represents a significant step toward unveiling the key molecular requirements to promote AMP expression in human intestinal epithelial cells, and revealing new potential targets for the development of an innovative therapeutic strategy aiming at stimulating the host AMP expression, at the era of antimicrobial resistance.

Exploring seasonal variations, assembly dynamics, and relationships of bacterial communities in different habitats of marine ranching.

Offshore coastal marine ranching ecosystems provide habitat for diverse and active bacterial communities. In this study, 16S rRNA gene sequencing and multiple bioinformatics methods were applied to investigate assembly dynamics and relationships in different habitats. The higher number of edges in the water network, more balanced ratio of positive and negative links, and more keystone species included in the co-occurrence network of water. Stochastic processes dominated in shaping gut and sediment community assembly (R2 < 0.5), while water bacterial community assembly were dominated by deterministic processes (R2 > 0.5). Dissimilarity-overlap curve model indicated that the communities in different habitats have general dynamics and interspecific interaction (P < 0.001). Bacterial source-tracking analysis revealed that the gut was more similar to the sediment than the water bacterial communities. In summary, this study provides basic data for the ecological study of marine ranching through the study of bacterial community dynamics.

Accelerated Charge Transfer through Interface Chemical Bonds in MoS2/TiO2 for Photocatalytic Conversion of Lignocellulosic Biomass to H2.

Solar photocatalytic H2 production from lignocellulosic biomass has attracted great interest, but it suffers from low photocatalytic efficiency owing to the absence of highly efficient photocatalysts. Herein, we designed and constructed ultrathin MoS2-modified porous TiO2 microspheres (MT) with abundant interface Ti-S bonds as photocatalysts for photocatalytic H2 generation from lignocellulosic biomass. Owing to the accelerated charge transfer related to Ti-S bonds, as well as the abundant active sites for both H2 and ●OH generation, respectively, related to the high exposed edge of MoS2 and the large specific surface area of TiO2, MT photocatalysts demonstrate good performance in the photocatalytic conversion of α-cellulose and lignocellulosic biomass to H2. The highest H2 generation rate of 849 µmol·g-1·h-1 and apparent quantum yield of 4.45% at 380 nm was achieved in α-cellulose aqueous solution for the optimized MT photocatalyst. More importantly, lignocellulosic biomass of corncob, rice hull, bamboo, polar wood chip, and wheat straw were successfully converted to H2 over MT photocatalysts with H2 generation rate of 10, 19, 36, 29, and 8 µmol·g-1·h-1, respectively. This work provides a guiding design approach to develop highly active photocatalysts via interface engineering for solar H2 production from lignocellulosic biomass.

Artemisinins ameliorate polycystic ovarian syndrome by mediating LONP1-CYP11A1 interaction.

Polycystic ovary syndrome (PCOS), a prevalent reproductive disorder in women of reproductive age, features androgen excess, ovulatory dysfunction, and polycystic ovaries. Despite its high prevalence, specific pharmacologic intervention for PCOS is challenging. In this study, we identified artemisinins as anti-PCOS agents. Our finding demonstrated the efficacy of artemisinin derivatives in alleviating PCOS symptoms in both rodent models and human patients, curbing hyperandrogenemia through suppression of ovarian androgen synthesis. Artemisinins promoted cytochrome P450 family 11 subfamily A member 1 (CYP11A1) protein degradation to block androgen overproduction. Mechanistically, artemisinins directly targeted lon peptidase 1 (LONP1), enhanced LONP1-CYP11A1 interaction, and facilitated LONP1-catalyzed CYP11A1 degradation. Overexpression of LONP1 replicated the androgen-lowering effect of artemisinins. Our data suggest that artemisinin application is a promising approach for treating PCOS and highlight the crucial role of the LONP1-CYP11A1 interaction in controlling hyperandrogenism and PCOS occurrence.

Dual-Mechanism Peptide SR25 has Broad Antimicrobial Activity and Potential Application for Healing Bacteria-infected Diabetic Wounds.

The rise of antibiotic resistance poses a significant public health crisis, particularly due to limited antimicrobial options for the treatment of infections with Gram-negative pathogens. Here, an antimicrobial peptide (AMP) SR25 is characterized, which effectively kills both Gram-negative and Gram-positive bacteria through a unique dual-targeting mechanism without detectable resistance. Meanwhile, an SR25-functionalized hydrogel is developed for the efficient treatment of infected diabetic wounds. SR25 is obtained through genome mining from an uncultured bovine enteric actinomycete named Nonomuraea Jilinensis sp. nov. Investigations reveal that SR25 has two independent cellular targets, disrupting bacterial membrane integrity and restraining the activity of succinate:quinone oxidoreductase (SQR). In a diabetic mice wound infection model, the SR25-incorporated hydrogel exhibits high efficacy against mixed infections of Escherichia coli (E. coli) and methicillin-resistant Staphylococcus aureus (MRSA), accelerating wound healing. Overall, these findings demonstrate the therapeutic potential of SR25 and highlight the value of mining drugs with multiple mechanisms from uncultured animal commensals for combating challenging bacterial pathogens.

A fine-grained reversible data hiding in encrypted domain based on the cipher-text redundancy of encryption process.

Considering the granularity of embedded data in the design of reversible data hiding scheme has important research significance for the permission control and management of multi-granularity information. To broaden the application possibilities of encrypted data in cloud environments, the researchers propose a fine-grained reversible data hiding method leveraging the cipher-text redundancy of ElGamal encryption. Initially, prior to the encryption process, pixels are organized into a full binary tree based on fine-grained access permissions. Subsequently, a chaotic sequence generator is employed to assign distinct embedding keys to each layer of the full binary tree according to the access permissions. Following this, an XOR operation is conducted between the embedding key and the corresponding secret information in each layer to derive the target features of the cipher-text, facilitating subsequent fine-grained data hiding. Throughout the ElGamal encryption process, iterative manipulation of the random variable ensures alignment between the cipher-text output and the target feature, enabling the embedding of secret information across different layers. This approach facilitates the fine-grained blind extraction of secret information from an encrypted state, thereby expanding the potential applications of cipher-text by extracting information without revealing the original data. Furthermore, the scheme enhances information security through distributed storage and conceals the presence of information hiding by leveraging the separability of lossless decryption and information extraction. Simulation results demonstrate that secret information of three granularities can be embedded and extracted without interference within a three-layer full binary structure, with a maximum embedding capacity of up to 1.75 bpp.

Oligomerization-mediated autoinhibition and cofactor binding of a plant NLR.

Nucleotide-binding leucine-rich repeat (NLR) proteins have a pivotal role in plant immunity by recognizing pathogen effectors1,2. Maintaining a balanced immune response is crucial, as excessive NLR expression can lead to unintended autoimmunity3,4. Unlike most NLRs, plant NLR required for cell death 2 (NRC2) belongs to a small NLR group characterized by constitutively high expression without self-activation5. The mechanisms underlying NRC2 autoinhibition and activation are not yet understood. Here we show that Solanum lycopersicum (tomato) NRC2 (SlNRC2) forms dimers and tetramers, and higher-order oligomers at elevated concentrations. Cryo-electron microscopy (cryo-EM) reveals an inactive conformation of SlNRC2 within these oligomers. Dimerization and oligomerization not only stabilize the inactive state but also sequester SlNRC2 from assembling into an active form. Mutations at the dimeric or inter-dimeric interfaces enhance pathogen-induced cell death and immunity in Nicotiana (N.) benthamiana. The cryo-EM structures unexpectedly reveal inositol hexakisphosphate (IP6) or pentakisphosphate (IP5) bound to the inner surface of SlNRC2's C-terminal LRR domain as confirmed by mass spectrometry. Mutations at the IP-binding site impair inositol phosphate binding of SlNRC2 and pathogen-induced SlNRC2-mediated cell death in N. benthamiana. Together, our study unveils a novel negative regulatory mechanism of NLR activation and suggests inositol phosphates as cofactors of NRCs.

Polyethyleneimine-mediated assembly of DNA nanotubes for KRAS siRNA delivery in lung adenocarcinoma therapy.

Self-assembled DNA nanostructures hold great promise in biosensing, drug delivery and nanomedicine. Nevertheless, challenges like instability and inefficiency in cellular uptake of DNA nanostructures under physiological conditions limit their practical use. To tackle these obstacles, this study proposes a novel approach that integrates the cationic polymer polyethyleneimine (PEI) with DNA self-assembly. The hypothesis is that the positively charged linear PEI can facilitate the self-assembly of DNA nanostructures, safeguard them against harsh conditions and impart them with the cellular penetration characteristic of PEI. As a demonstration, a DNA nanotube (PNT) was successfully synthesized through PEI mediation, and it exhibited significantly enhanced stability and cellular uptake efficiency compared to conventional Mg2+-assembled DNA nanotubes. The internalization mechanism was further found to be both clathrin-mediated and caveolin-mediated endocytosis, influenced by both PEI and DNA. To showcase the applicability of this hybrid nanostructure for biomedical settings, the KRAS siRNA-loaded PNT was efficiently delivered into lung adenocarcinoma cells, leading to excellent anticancer effects in vitro. These findings suggest that the PEI-mediated DNA assembly could become a valuable tool for future biomedical applications.

USP36 inhibits apoptosis by deubiquitinating cIAP1 and survivin in colorectal cancer cells.

Chemotherapeutic agents for treating colorectal cancer (CRC) primarily induce apoptosis in tumor cells. The ubiquitin-proteasome system is critical for apoptosis regulation. Deubiquitinating enzymes (DUBs) remove ubiquitin from substrates to reverse ubiquitination. Although over 100 DUB members have been discovered, the biological functions of only a small proportion of DUBs have been characterized. Here, we aimed to systematically identify the DUBs that contribute to the development of CRC. Among the DUBs, ubiquitin-specific protease 36 (USP36) is upregulated in CRC. We showed that the knockdown of USP36 induces intrinsic and extrinsic apoptosis. Through gene silencing and coimmunoprecipitation techniques, we identified survivin and cIAP1 as USP36 targets. Mechanistically, USP36 binds and removes lysine-11-linked ubiquitin chains from cIAP1 and lysine-48-linked ubiquitin chains from survivin to abolish protein degradation. Overexpression of USP36 disrupts the formation of the XIAP-second mitochondria-derived activator of caspase complex and promotes receptor-interacting protein kinase 1 ubiquitination, validating USP36 as an inhibitor to intrinsic and extrinsic apoptosis through deubiquitinating survivin and cIAP1. Therefore, our results suggest that USP36 is involved in CRC progression and is a potential therapeutic target.

Circular economy approach: Nutrient recovery and economical struvite production from wastewater sources by using modified biochars.

The enrichment of phosphorus (P) and nitrogen (N) in aquatic systems can cause eutrophication. Moreover, P rocks may become exhausted in the next 100 years. A slow-release fertilizer called struvite (MgNH4PO4.6H2O) can reduce surface runoff. However, the high cost of raw material or chemicals is a bottleneck in their economical production. Therefore, incinerated sewage sludge ash, food wastewater, and bittern were combined as the sources of P, N, and Mg, respectively. Sawdust biochar was used to enhance the adsorptive recovery of nutrients. First, recovery kinetics was studied by comparing bittern-impregnated biochar (BtB) with the Mg-impregnated biochar (MgB). Subsequently, the synergistic physical and chemical interactions were observed for P and N recovery. Almost complete PO43-P recoveries were achieved within 10 min for both biochars. However, NH4+-N recovery was stable after 2 h, with 26% recovery by MgB and 20% recovery by BtB. Biochars activated with steam (steam-activated biochar) and KOH (KOH-activated biochar) gave superior activities to those of unactivated biochars and activated carbon (AC) nutrient recovery and struvite purity. Moreover, the activated biochars showed a lower risk of surface runoff, similar to that of AC. Therefore, activated biochars can be used as an alternative to AC for economical struvite production from a combination of wastewater sources.