Genome-wide identification and characterization of DTX family genes highlighting their locations, functions, and regulatory factors in banana (Musa acuminata).

The detoxification efflux carriers (DTX) are a significant group of multidrug efflux transporter family members that play diverse functions in all kingdoms of living organisms. However, genome-wide identification and characterization of DTX family transporters have not yet been performed in banana, despite its importance as an economic fruit plant. Therefore, a detailed genome-wide analysis of DTX family transporters in banana (Musa acuminata) was conducted using integrated bioinformatics and systems biology approaches. In this study, a total of 37 DTX transporters were identified in the banana genome and divided into four groups (I, II, III, and IV) based on phylogenetic analysis. The gene structures, as well as their proteins' domains and motifs, were found to be significantly conserved. Gene ontology (GO) annotation revealed that the predicted DTX genes might play a vital role in protecting cells and membrane-bound organelles through detoxification mechanisms and the removal of drug molecules from banana cells. Gene regulatory analyses identified key transcription factors (TFs), cis-acting elements, and post-transcriptional regulators (miRNAs) of DTX genes, suggesting their potential roles in banana. Furthermore, the changes in gene expression levels due to pathogenic infections and non-living factor indicate that banana DTX genes play a role in responses to both biotic and abiotic stresses. The results of this study could serve as valuable tools to improve banana quality by protecting them from a range of environmental stresses.

Future Industrial Applications: Exploring LPWAN-Driven IoT Protocols.

The Internet of Things (IoT) will bring about the next industrial revolution in Industry 4.0. The communication aspect of IoT devices is one of the most critical factors in choosing the device that is suitable for use. Thus far, the IoT physical layer communication challenges have been met with various communications protocols that provide varying strengths and weaknesses. This paper summarizes the network architectures of some of the most popular IoT wireless communications protocols. It also presents a comparative analysis of some of the critical features, including power consumption, coverage, data rate, security, cost, and quality of service (QoS). This comparative study shows that low-power wide area network (LPWAN)-based IoT protocols (LoRa, Sigfox, NB-IoT, LTE-M) are more suitable for future industrial applications because of their energy efficiency, high coverage, and cost efficiency. In addition, the study also presents an Industrial Internet of Things (IIoT) application perspective on the suitability of LPWAN protocols in a particular scenario and addresses some open issues that need to be researched. Thus, this study can assist in deciding the most suitable IoT communication protocol for an industrial and production field.

Prevalence of microplastics in commonly consumed fish species of the river Old Brahmaputra, Bangladesh.

Microplastics (MPs) are pervasive in aquatic environments, but inland waterbodies (rivers and floodplains) have received much less attention. The present study assesses the incidence of MPs in the gastrointestinal tracts of five commercially important edible fish species-two column feeders (n = 30) and three benthivores (n = 45) from upstream, midstream, and downstream of the Old Brahmaputra river in north-central Bangladesh. MPs were detected in 58.93% of fish, with the highest level in freshwater eel, Mastacembelus armatus (10.31 ± 0.75/fish). Fibers (49.03%) and pellets (28.02%) were the most frequent MPs. Nearly 72% MPs were smaller than 1 mm, and 50.97% were black. FTIR analysis showed 59% polyethelene (PE), followed by polyamide (40%) and unidentified (1%). MP ingestion was linked to fish size and weight, and a high incidence was recorded in the downstream river. Two omnivorous benthic fish ingest more MPs than others. The results corroborate the presence of MPs in the inland river and fish fauna and augment our understanding of heterogeneous MP uptake by fish.

Triptycene-Fused Sterically Shielded Multi-Resonance TADF Emitter Enables High-Efficiency Deep Blue OLEDs with Reduced Dexter Energy Transfer.

Designing multi-resonance (MR) emitters that can simultaneously achieve narrowband emission and suppressed intermolecular interactions is challenging for realizing high color purity and stable blue organic light-emitting diodes (OLEDs). Herein, a sterically shielded yet extremely rigid emitter based on a triptycene-fused B,N core (Tp-DABNA) is proposed to address the issue. Tp-DABNA exhibits intense deep blue emissions with a narrow full width at half maximum (FWHM) and a high horizontal transition dipole ratio, superior to the well-known bulky emitter, t-DABNA. The rigid MR skeleton of Tp-DABNA suppresses structural relaxation in the excited state, with reduced contributions from the medium- and high-frequency vibrational modes to spectral broadening. The hyperfluorescence (HF) film composed of a sensitizer and Tp-DABNA shows reduced Dexter energy transfer compared to those of t-DABNA and DABNA-1. Notably, deep blue TADF-OLEDs with the Tp-DABNA emitter display higher external quantum efficiencies (EQEmax =24.8 %) and narrower FWHMs (≤26 nm) than t-DABNA-based OLEDs (EQEmax =19.8 %). The HF-OLEDs based on the Tp-DABNA emitter further demonstrate improved performance with an EQEmax of 28.7 % and mitigated efficiency roll-offs.

Biological Interfacial Materials for Organic Light-Emitting Diodes.

Organic optoelectronic devices have received appreciable attention due to their low cost, mechanical flexibility, band-gap engineering, lightness, and solution processability over a broad area. Specifically, realizing sustainability in organic optoelectronics, especially in solar cells and light-emitting devices, is a crucial milestone in the evolution of green electronics. Recently, the utilization of biological materials has appeared as an efficient means to alter the interfacial properties, and hence improve the performance, lifetime and stability of organic light-emitting diodes (OLEDs). Biological materials can be known as essential renewable bio-resources obtained from plants, animals and microorganisms. The application of biological interfacial materials (BIMs) in OLEDs is still in its early phase compared to the conventional synthetic interfacial materials; however, their fascinating features (such as their eco-friendly nature, biodegradability, easy modification, sustainability, biocompatibility, versatile structures, proton conductivity and rich functional groups) are compelling researchers around the world to construct innovative devices with enhanced efficiency. In this regard, we provide an extensive review of BIMs and their significance in the evolution of next-generation OLED devices. We highlight the electrical and physical properties of different BIMs, and address how such characteristics have been recently exploited to make efficient OLED devices. Biological materials such as ampicillin, deoxyribonucleic acid (DNA), nucleobases (NBs) and lignin derivatives have demonstrated significant potential as hole/electron transport layers as well as hole/electron blocking layers for OLED devices. Biological materials capable of generating a strong interfacial dipole can be considered as a promising prospect for alternative interlayer materials for OLED applications.

Integrated transcriptome catalog of Tenualosa ilisha as a resource for gene discovery and expression profiling.

The silver pride of Bangladesh, migratory shad, Tenualosa ilisha (Hilsa), makes the highest contribution to the total fish production of Bangladesh. Despite its noteworthy contribution, a well-annotated transcriptome data is not available. Here we report a transcriptomic catalog of Hilsa, constructed by assembling RNA-Seq reads from different tissues of the fish including brain, gill, kidney, liver, and muscle. Hilsa fish were collected from different aquatic habitats (fresh, brackish, and sea water) and the sequencing was performed in the next generation sequencing (NGS) platform. De novo assembly of the sequences obtained from 46 cDNA libraries revealed 462,085 transcript isoforms that were subsequently annotated using the Universal Protein Resource Knowledgebase (UniPortKB) as a reference. Starting from the sampling to final annotation, all the steps along with the workflow are reported here. This study will provide a significant resource for ongoing and future research on Hilsa for transcriptome based expression profiling and identification of candidate genes.

YCl3-Substituted CsPbI3 Perovskite Nanorods for Efficient Red-Light-Emitting Diodes.

Cesium lead iodide (CsPbI3) perovskite nanocrystals (NCs) are a promising material for red-light-emitting diodes (LEDs) due to their excellent color purity and high luminous efficiency. However, small-sized CsPbI3 colloidal NCs, such as nanocubes, used in LEDs suffer from confinement effects, negatively impacting their photoluminescence quantum yield (PLQY) and overall efficiency. Here, we introduced YCl3 into the CsPbI3 perovskite, which formed anisotropic, one-dimensional (1D) nanorods. This was achieved by taking advantage of the difference in bond energies among iodide and chloride ions, which caused YCl3 to promote the anisotropic growth of CsPbI3 NCs. The addition of YCl3 significantly improved the PLQY by passivating nonradiative recombination rates. The resulting YCl3-substituted CsPbI3 nanorods were applied to the emissive layer in LEDs, and we achieved an external quantum efficiency of ~3.16%, which is 1.86-fold higher than the pristine CsPbI3 NCs (1.69%) based LED. Notably, the ratio of horizontal transition dipole moments (TDMs) in the anisotropic YCl3:CsPbI3 nanorods was found to be 75%, which is higher than the isotropically-oriented TDMs in CsPbI3 nanocrystals (67%). This increased the TDM ratio and led to higher light outcoupling efficiency in nanorod-based LEDs. Overall, the results suggest that YCl3-substituted CsPbI3 nanorods could be promising for achieving high-performance perovskite LEDs.

Eculizumab in the Treatment of Gemcitabine-Induced Atypical Hemolytic Uremic Syndrome.

Gemcitabine-induced hemolytic uremic syndrome is an often-missed condition. We present a case outlining the successful management of a patient with metastatic cholangiocarcinoma treated with gemcitabine who subsequently developed hemolytic uremic syndrome. Early recognition and stopping gemcitabine are essential in this patient population. Complement inhibitors have been used, and our patient improved on eculizumab therapy.

Identification of SARS CoV-2 Omicron BA.1 and a novel Delta lineage by rapid methods and partial spike protein sequences in Sulaymaniyah Province, Iraq.

BACKGROUND: Five variants of concern (VOCs) of severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) have been globally recorded including Alpha, Beta, Gamma, Delta, and Omicron. The Omicron variant has outcompeted the other variants including the Delta variant. Molecular screenings of VOCs are important for surveillance, treatment, and vaccination programs. This study aimed to identify VOCs by using rapid inexpensive methods and partial sequencing of the virus's spike gene. METHODS: Mutation-specific rRT PCR probes were used for both D614G and K417N mutations to potentially discriminate between Delta and Omicron variants. These were followed by sequencing of a fragment of spike gene (748 nucleotides), which covers the most notable VOC mutations in the receptor binding domain of SARS CoV-2. RESULTS: Rapid methods showed that out of 24 SARS CoV-2 positive samples, 19 carried the N417 mutation, which is present in the Omicron variant. Furthermore, 3 samples carried K417 wildtype, which is present in the Delta variant. Additionally, 2 samples containing both K417 and N417 suggested mixed infections between the two variants. The D614G mutation was present in all samples. Among the 4 samples sequenced, 3 samples carried 13 mutations, which are present in Omicron BA.1. The fourth sample contained the two common mutations (T478K and L452R) present in Delta, in addition to two more rare mutations (F456L and F490S), which are not commonly seen in Delta. Our data suggested that both Omicron variant BA.1 and a novel Delta variant might have circulated in this region that needs further investigations.

Physicochemical and Nutritional Characteristics of Cookies Prepared with Untapped Seaweed Ulva intestinalis: An Approach to Value Addition as a Functional Food.

The present study was investigated to know the sensory, physicochemical, nutritional and fatty acid properties of seaweed-based cookies prepared with untapped seaweed Ulva intestinalis (UI) from Bangladesh coast. The cookies were formulated with different percentages of UI inclusions both in powdered (PUI) and fragmented (FUI) forms, in order to evaluate different quality attributes in prepared value-added cookies. In sensory analysis, seaweed inclusion levels of 1% PUI, 2.5% PUI, 1% FUI, 2.5% FUI and 5% FUI to cookies were acceptable by panelists. Considering the maximum percentage of seaweed inclusions, 2.5% PUI and 5% FUI were selected for further analysis. The results of physicochemical properties such as moisture content, spread factor, baking loss, pH, cookie density, color, texture properties, volatile basic nitrogen and thiobarbituric acid reactive species were within acceptable limits. In nutritional analysis, 2.5% PUI and 5% FUI cookies showed a remarkable and significant increase in lipid and ash contents, compared to untreated controls. Being the first report on fatty acids profile by UI from Bangladesh, among 24 fatty acids identified, the amount of total saturated, mono-unsaturated, omega-3 fatty acids and omega-6 fatty acids were reported to be 641.9 (36.2%), 563.7 (31.8%), 133.8 (7.6%) and 436.3 (24.6%) µg/g DW, respectively. The results suggest that cookies with 2.5% PUI and 5% FUI can be marketed as healthy foods for consumers.

A finite element study of a fractured tibia treated with a unilateral external fixator: The effects of the number of pins and cortical thickness.

INTRODUCTION: In the early stage of fracture fixation, the aim of a unilateral external fixator (UEF) to stimulate healing and maintain stability may be suppressed by using inadequate number of pins. Cortical thinning due to age or osteoporosis endangers a successful fracture fixation. MATERIALS AND METHODS: This study evaluates the initial strength and stability of the fracture fixation and tissue differentiation under the influences of variable cortical thickness (5 mm to 1 mm) and variable number of pins (1 to 4 in each bone fragment). A finite element program was utilised to develop 20 three-dimensional models of simplified diaphyseal tibia with fracture callus fixed with UEF. A mechano-regulation code based on the deviatoric strain theory was written and applied to simulate tissue differentiation. The values of von Mises stress, interfragmentary strain (IFS), and fibrocartilage index (FCI) were evaluated. RESULTS: Cortical thinning from 5 mm to 1 mm increased IFS and FCI by an average of 30.3% and 18.7%, respectively, and resulted in higher stresses in the UEF and bone. Using 1 pin in each bone fragment produced excessive IFS in the models with 1 mm, 2 mm and 3 mm cortical thickness. Inserting the second pin into the bone fragment could considerably reduce the IFS and fibrocartilaginous tissue formation in the fracture site and improve load transmission to the fixator. Whereas inserting the fourth pin could minimally affect the mechano-biological environment of healing. CONCLUSIONS: This study suggests that initial instability due to cortical thinning can be efficiently alleviated by adding the number of pins up to 3 in a UEF; additionally, it may improve the knowledge about applying UEFs adequately stable, whilst promoting inclination toward endochondral ossification, simultaneously.

Antibiotic susceptibility pattern of fish pathogens: A new approach of emerging the bacterial resistance through biofilm formation in in-vitro condition.

BACKGROUND: The ability of many bacteria to adhere on the host surfaces and forming biofilms has major implications in a wide variety of industries including the food industry, where biofilms may create a persistent source of contamination. In the same environmental condition, the multiple bacterial species can closely interact with each other and may easily enhance their drug resistance capability, which finally increases the multi-drug resistant (MDR) attribute of the species. OBJECTIVE: The present study examined whether the mixed-species biofilm possesses any impact on the enhancement of the antibiotic resistance of the planktonic or single-cell bacterial isolates present in the fish samples. METHODS: In this regard, Cyprinus rubrofuscus (Koi), Heteropneustes fossilis (Shing) and Mystus vittatus (Tengra) fishes were collected and subjected to form an in vitro biofilm by shaking condition into the wise bath. The drug-resistant pattern was determined by the Kirby Bauer technique. RESULTS: All the samples exhibited a huge array (up to 107 cfu/ml or g) of bacteria such as E. coli, Klebsiella spp., Vibrio spp., Salmonella spp., Proteus spp. and Staphylococcus spp. The isolates from both the bulk samples and their corresponding biofilms were subjected to antibiogram assay using antibiotics such as Ampicillin (10 µg), Erythromycin (15 µg), Streptomycin (STP 10 µg), Oxacillin (10 µg), Nalidixic acid (30 µg). Before biofilm formation, few of the isolates were found to be sensitive and few were resistant against the antibiotics. But when the species were isolated from the biofilm the sensitive one acquired drug resistance and resistant strain unveiled more resistance towards the same antibiotics. The present study revealed extensive bacterial contamination in fish samples among those some were resistant against the supplied drugs. CONCLUSION: After the formation of multi-species biofilm, the isolates became more resistant against the same drugs that is alarming for consumers and major obstacles to maintain sustainable health.

Computational and experimental insight into antituberculosis agent, (E)-benzyl-2-(4-hydroxy-2-methoxybenzylidene) hydrazinecarbodithioate: ADME analysis.

A new Schiff base, (E)-benzyl-2-(4-hydroxy-2-methoxybenzylidene)hydrazinecarbodithioate (compound 1) has been synthesized and experimentally characterized by the IR, UV-Vis, 1H-NMR and mass spectroscopies. The theoretical study of the synthesized compound was evaluated using the density functional theory (DFT) at B3LYP/6-31G+(d,p) basis set. The electronic absorption spectrum of compound 1 was evaluated using time-dependent density functional theory. Besides, in silico studies were done for the prediction of absorption, distribution, metabolism and excretion profiles of compound 1. According to the result, the theoretical data were well fitted with the experimental values. The studied compound has low chemical reactivity and high kinetic stability. In the molecular electrostatic potential map, the negative and positive potential sites were found around electronegative atoms and hydrogen atoms of compound 1, respectively. The 97.75% Lewis and 2.25% non-Lewis structure were present in the studied molecule. The molecular docking results reveal that compound 1 can be used as antituberculosis agent as compare to ethambutol.

Plant growth promoting endophyte Burkholderia contaminans NZ antagonizes phytopathogen Macrophomina phaseolina through melanin synthesis and pyrrolnitrin inhibition.

The endophytic bacterium Burkholderia contaminans NZ was isolated from jute, which is an important fiber-producing plant. This bacterium exhibits significant growth promotion activity in in vivo pot experiments, and like other plant growth-promoting (PGP) bacteria fixes nitrogen, produces indole acetic acid (IAA), siderophore, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. B. contaminans NZ is considered to exert a promising growth inhibitory effect on Macrophomina phaseolina, a phytopathogen responsible for infecting hundreds of crops worldwide. This study aimed to identify the possibility of B. contaminans NZ as a safe biocontrol agent and assess its effectiveness in suppressing phytopathogenic fungi, especially M. phaseolina. Co-culture of M. phaseolina with B. contaminans NZ on both solid and liquid media revealed appreciable growth suppression of M. phaseolina and its chromogenic aberration in liquid culture. Genome mining of B. contaminans NZ using NaPDoS and antiSMASH revealed gene clusters that displayed 100% similarity for cytotoxic and antifungal substances, such as pyrrolnitrin. GC-MS analysis of B. contaminans NZ culture extracts revealed various bioactive compounds, including catechol; 9,10-dihydro-12'-hydroxy-2'-methyl-5'-(phenylmethyl)- ergotaman 3',6',18-trione; 2,3-dihydro-3,5- dihydroxy-6-methyl-4H-pyran-4-one; 1-(1,6-Dioxooctadecyl)- pyrrolidine; 9-Octadecenamide; and 2- methoxy- phenol. These compounds reportedly exhibit tyrosinase inhibitory, antifungal, and antibiotic activities. Using a more targeted approach, an RP-HPLC purified fraction was analyzed by LC-MS, confirming the existence of pyrrolnitrin in the B. contaminans NZ extract. Secondary metabolites, such as catechol and ergotaman, have been predicted to inhibit melanin synthesis in M. phaseolina. Thus, B. contaminans NZ appears to inhibit phytopathogens by apparently impairing melanin synthesis and other potential biochemical pathways, exhibiting considerable fungistatic activity.

SARS-CoV-2 vaccines breakthrough infection hospitalizations after one dose in Libya: cohort study

SARS-CoV-2 infection is widely spread over people, from youth to the elderly. Vaccination against SARS-CoV-2 is an important preventive measurement to help end the SARS-CoV-2 pandemic. From 30 April to 15 July, we collected the number of people infected with SARS-COV-2 and the mortality rate from daily reports issued by the National Center for Disease Control of Libya (NCDC). Approximately 445000 doses have been administered in Libya since 10 April, and 5 thousand doses are now being administered during this period on a daily basis. To estimate the rate of breakthrough vaccine infection of the SARS-COV-2 in Libya. We found that one dose of the three different types of vaccines had decreased the virus transmission across people and mortality rate until 10 weeks after the first dose. This study highlights the dramatic success of the early months of the nations coronavirus vaccines rollout.

Targeting SARS-CoV-2 nonstructural protein 15 endoribonuclease: an in silico perspective.

The newly emerged human coronavirus, SARS-CoV-2, had begun to spread last year and sparked worldwide. In this study, molecular docking is utilized to test some previously approved drugs against the SARS-CoV-2 nonstructural protein 15 (Nsp15). We screened 23 drugs, from which three (saquinavir, valrubicin and aprepitant) show a paramount predicted binding affinity (-9.1, -9.6 and -9.2 kcal/mol, respectively) against SARS-CoV-2 Nsp15. Moreover, saquinavir and aprepitant make nonbonded interactions with Leu201 in the active site cavity of Nsp15, while the drug valrubicin interacts with Arg199 and Leu201. This binding pattern may be effective against the targeted protein, leading to Nsp15 blockage and virus abolition. Additionally, the pharmacological properties of the screened drugs are known since they have been approved against different viruses.

In Silico Characterization of a Hypothetical Protein from Shigella dysenteriae ATCC 12039 Reveals a Pathogenesis-Related Protein of the Type-VI Secretion System.

Shigellosis caused by Shigella dysenteriae is a major public health concern worldwide, particularly in developing countries. The bacterial genome is known, but there are many hypothetical proteins whose functions are yet to be discovered. A hypothetical protein (accession no. WP_128879999.1, 161 residues) of S. dysenteriae ATCC 12039 strain was selected in this study for comprehensive structural and functional analysis. Subcellular localization and different physicochemical properties of this hypothetical protein were estimated indicating it as a stable, soluble, and extracellular protein. Functional annotation tools, such as NCBI-CD Search, Pfam, and InterProScan, predicted our target protein to be an amidase effector protein 4 (Tae4) of type-VI secretion system (T6SS). Multiple sequence alignment of the homologous sequences coincided with previous findings. Random coil was found to be predominant in secondary structure. Three-dimensional (3D) structure of the protein was obtained using homology modeling method by SWISS-MODEL server using a template protein (PDB ID: 4J30) of 80.12% sequence identity. The 3D structure became more stable after YASARA energy minimization and was validated by several quality assessment tools like PROCHECK, QMEAN, Verify3D, and ERRAT. Superimposition of the target with the template protein by UCSF Chimera generated RMSD value of 0.115 Å, suggesting a reliable 3D structure. The active site of the modeled structure was predicted and visualized by CASTp server and PyMOL. Interestingly, similar binding affinity and key interacting residues were found for the target protein and a Salmonella enterica Tae4 protein with the ligand L-Ala D-Glu-mDAP by molecular docking analysis. Protein-protein docking was also performed between the target protein and hemolysin coregulated protein 1 of T6SS. Finally, the protein was found to be a unique protein of S. dysenteriae nonhomologous to human by comparative genomics approach indicating a potential therapeutic target. Most pathogens harboring T6SS in their system pose a significant threat to the human health. Many T6SSs and their effectors are associated with interbacterial competition, pathogenesis, and virulency; however, relationships between these effectors and pathogenicity of S. dysenteriae are yet to be determined. The study findings provide a lucrative platform for future antibacterial treatment.

A plant endophyte Staphylococcus hominis strain MBL_AB63 produces a novel lantibiotic, homicorcin and a position one variant.

Here we report a jute endophyte Staphylococcus hominis strain MBL_AB63 isolated from jute seeds which showed promising antimicrobial activity against Staphylococcus aureus SG511 when screening for antimicrobial substances. The whole genome sequence of this strain, annotated using BAGEL4 and antiSMASH 5.0 to predict the gene clusters for antimicrobial substances identified a novel antimicrobial peptide cluster that belongs to the class I lantibiotic group. The predicted lantibiotic (homicorcin) was found to be 82% similar to a reported peptide epicidin 280 having a difference of seven amino acids at several positions of the core peptide. Two distinct peaks obtained at close retention times from a RP-HPLC purified fraction have comparable antimicrobial activities and LC-MS revealed the molecular mass of these peaks to be 3046.5 and 3043.2 Da. The presence of an oxidoreductase (homO) similar to that of epicidin 280- associated eciO or epilancin 15X- associated elxO in the homicorcin gene cluster is predicted to be responsible for the reduction of the first dehydrated residue dehydroalanine (Dha) to 2-hydroxypropionate that causes an increase of 3 Da mass of homicorcin 1. Trypsin digestion of the core peptide and its variant followed by ESI-MS analysis suggests the presence of three ring structures, one in the N-terminal and other two interlocking rings at the C-terminal region that remain undigested. Homicorcin exerts bactericidal activity against susceptible cells by disrupting the integrity of the cytoplasmic membrane through pore formation as observed under FE-SEM.

Diagnostic Utility and Significance of Performing Multistep Level Sections in Breast and Gynecologic Biopsy Specimens.

OBJECTIVES: The significance of performing multistep level sections, including preparation of unstained sections in breast and gynecologic biopsy specimens, has been studied. METHODS: Consecutive H&E-stained level sections of 785 atypical and malignant biopsy specimens were included. The diagnostic material was categorized into present, absent, increased in size, or depleted. If the multistep level sections helped in establishing the diagnosis after a nondiagnostic material or the tissue significantly increased in size, this was considered a positive impact. RESULTS: No effect and positive impact of performing multistep level sections were obtained in 84.8% and 15.2% by preparing a second level and 97.2% and 2.8% by preparing a third level, respectively. Eighteen (2.3%) of the diagnoses could have been missed without performing a second level, while 8 (1%) could have been missed without performing a third level. The intervening unstained sections were used in 27 of 785 (3.4%) of the cases. CONCLUSIONS: Staining two level sections with H&E significantly affected the diagnosis. However, preparing a third level did not improve the diagnosis. A universal protocol should be considered to standardize the handling of biopsy specimens among laboratories.