Genotyping single nucleotide polymorphisms in homologous regions using multiplex kb level amplicon capture sequencing.

Single nucleotide polymorphisms (SNPs) in homologous regions play a critical role in the field of genetics. However, genotyping these SNPs is challenging due to the presence of repetitive sequences within genome, which demand specific method. We introduce a new, mid-throughput method that simplifies SNP genotyping in homologous DNA sequences by utilizing a combination of multiplex kb level PCR (PCR size 2.5k-3.5 kb) for capturing targeted regions and multiplex nested PCR library construction for next-generation sequencing (Multi-kb level capture-seq). First of all, we randomly selected 7 SNPs in homologous regions and successfully captured 6-plex kb level amplicons (one of segments contains 2 SNPs, while the remaining segments each have only one SNP) in a single tube. And then, the amplification products were subjected to multiplex nested PCR for library construction and sequenced on Illumina platform. We tested this strategy using 600 amplicons from 100 samples and accurately genotyped 96.8% of target SNPs with a coverage depth of ≥ 15×. For the uniformity within the samples, over 66.7% (4/6) of the amplicons had a coverage depth above 0.2-fold of average sequencing depth. To validate the accuracy of this approach, we performed Ligase detection reaction PCR for genotyping the 100 samples, and found that the genotyping data was 97.71% consistent with our NGS results. In conclusion, we have developed a highly efficient and accurate method for SNP genotyping in homologous regions, which offers researchers a new strategy to explore the complex regions of genome.

Low cycle number multiplex PCR: A novel strategy for the construction of amplicon libraries for next-generation sequencing.

Multiplex PCR is a critical step when preparing amplicon library for next-generation sequencing. However, there are several challenges related to multiplex PCR including poor uniformity, nonspecific amplification, and primer-dimers. To address these issues, we propose a novel solution strategy that involves using a low cycle number (<10 cycles) in multiplex PCR and then employing carrier DNAs and magnetic beads for the selection of targeted products. This technique improves the amplicon uniformity while also reducing primer-dimers and PCR artifacts. To evaluate our technique, we initially utilized 120 DNA fragments from mouse genome containing single nucleotide polymorphism (SNP) sites. Sequencing results demonstrated that with only 7 cycles of multiplex PCR, 95.8% of the targeted SNP sites were mapped, with a coverage of at least 1×. The average sequencing depth of all amplicons was 1705.79 ± 1205.30×; 87% of them reached a coverage depth that exceeded 0.2-fold of the average sequencing depth. Our method had a greater uniformity (87%) when compared to Hi-Plex PCR (53.3%). Furthermore, we validated our strategy by randomly selecting 90 primer pairs twice from the initial set of 120 primer-pairs. Next, we used the same protocol to prepare amplicon libraries. The two groups had an average sequencing depth of 1013.30 ± 585.57× and 219.10 ± 158.27×, respectively; over 84% of the amplicons had a sequencing depth that exceeded 0.2-fold of average depth. These results suggest that the use of a low cycle number in multiplex PCR is a cost-effective and efficient approach for the preparation of amplicon libraries.

Microglia play an important role in PRV infection-induced immune responses of the central nervous system.

Pseudorabies virus (PRV) can infect multiple hosts and lead to fatal encephalitis. There is a significant increase in the number of microglia in the brain of animals infected with PRV. However, whether and how microglia contribute to central nervous system damage in PRV infection remain unknown. In the present study, we elucidated that PRV infection can cause more severe inflammatory cell infiltration, thicker and more numerous vessel sleeve walls, and more severe inflammatory responses in the brains of natural hosts (pigs) than in those of nonnatural hosts (mice). In a mice infection model, activated microglia restricted viral replication in the early stage of infection. Acute neuroinflammation caused by microglia hyperactivation at late-stage of infection. Furthermore, in vitro experiments revealed that microglia restricted viral replication and decreased viral infectivity. This may be associated with the phagocytic ability of microglia because we observed a significant increase in the expression of the membrane receptor TREM2 in microglia, which is closely related to phagocytosis, we observed that depletion of microglia exacerbated neurological symptoms, blood-brain barrier breakdown, and peripheral lymphocyte infiltration. Taken together, we revealed the dual role of microglia in protecting the host and neurons from PRV infection.

Generation, mechanisms, kinetics, and effects of gaseous chlorine dioxide in food preservation.

Food preservation is a critical issue in ensuring food safety and quality. Growing concern around industrial pollution of food and demand for environmentally sustainable food has led to increased interest in developing effective and eco-friendly preservation techniques. Gaseous ClO2 has gained attention for its strong oxidizing properties, high efficacy in microorganism inactivation, and potential for preserving the attributes and nutritional quality of fresh food while avoiding the formation of toxic byproducts or unacceptable levels of residues. However, the widespread use of gaseous ClO2 in the food industry is limited by several challenges. These include large-scale generation, high cost and environmental considerations, a lack of understanding of its mechanism of action, and the need for mathematical models to predict inactivation kinetics. This review aims to provide an overview of the up-to-date research and application of gaseous ClO2 . It covers preparation methods, preservation mechanisms, and kinetic models that predict the sterilizing efficacy of gaseous ClO2 under different conditions. The impacts of gaseous ClO2 on the quality attributes of fresh produce and low-moisture foods, such as seeds, sprouts, and spices, are also summarized. Overall, gaseous ClO2 is a promising preservation approach, and future studies are needed to address the challenges in large-scale generation and environmental considerations and to develop standardized protocols and databases for safe and effective use in the food industry.

When Fluorescent Sensing Meets Electrochemical Amplifying: A Powerful Platform for Gene Detection with High Sensitivity and Specificity.

Ultrasensitive and ultraselective detection of the gene requires emergency development to meet the medical demands and infectious disease control. Herein we report a versatile and scalable method based on electrochemical-chemical-cyclic amplification (EC-CA) and fluorescence detection for ultrasensitive gene sensing. The EC-CA is achieved by an electro-Fenton reaction (EFR). The hydroxyl radicals generated at EFR are trapped by terephthalic acid to form highly fluorescent 2-hydroxyterephthalic acid, which can be sensitively detected by a fluorescence spectrophotometer. The method is the first to be able to amplify the signal and reduce the noise simultaneously by using the conventional analytical methods directly. This described method can be used for reliable Fe3+ quantification in the range from 0.1 nM to 0.08 mM. The calculated limit of detection (LOD) is 0.02 nM. Then, hepatitis B virus (HBV) and p53 gene were detected by this proposed method through introducing the Fe3O4 nanoparticles into the gene hybridization system. The LODs for HBV and p53 gene even topped out at 2.6 pM and 1.7 fM, respectively. We demonstrated that the finally recorded signal was triply amplified through the EC cycle, Fe3O4 nanoparticles, and sensitive fluorescence detection. At the same time, the background signal arisen from matrix effects and readout noise was effectively suppressed. This method shows it is simple, convenient, and operational through the detection of Fe3+, HBV, and the p53 gene in blood samples, respectively. We believe our method will make a significant, near-term impact on the development of high-sensitivity methods that are versatile and scalable.

Electrochemical determination of artemisinin based on signal inhibition for the reduction of hemin.

A novel electrochemical sensor was constructed for the determination of artemisinin (ART) based on the inhibition of redox for hemin caused by ART. As far as we know, this strategy for ART determination may be proposed for the first time. In this work, untreated multi-walled carbon nanotubes were cast on the glassy carbon electrode (GCE) as conductive carrier. We prepared a bimetallic organic framework named FeGd-MOF and combined it with hemin by a simple physical mixed method. Then, we fabricated the working electrode by layer-by-layer modification and immobilization. The sensor measured by the differential pulse voltammetry (DPV) technique had calibration curves for the determination of ART, which was 0.3-350 µM with the correlation coefficient R2 = 0.9998. Furthermore, the obtained linear range could be practically used in real sample analysis such as dried leaves of Artemisia apiacea. Under the optimized condition, the electrochemical sensor exhibited high sensitivity, good stability, and excellent anti-interference performance. The limit of detection (LOD) for this sensor was 0.17 µM (signal to noise ratio, S/N = 3), which was much lower than that for some other reported electrochemical sensors. The recovery rates were in the range of 99.54-104.34% in real samples, indicating that the sensor had good repetition and high accuracy. Graphical abstract.

The Potential of Gaseous Chlorine Dioxide for the Control of Citrus Postharvest Stem-End Rot Caused by Lasiodiplodia theobromae.

The focus of this study was to develop technologies using chlorine dioxide (ClO2) gas to control postharvest stem-end rot of citrus caused by Lasiodiplodia theobromae. Mycelial growth of L. theobromae on potato dextrose agar (PDA) plugs was completely inhibited by a 24-h ClO2 exposure provided by 0.5 g of solid ClO2 generating granular mixture in a 7.7-liter sealed container. In vivo experiments were conducted on artificially inoculated Tango and naturally infected U.S. Early Pride mandarins. When ClO2 treatments were initiated 0 to 6 h after inoculation, decay development was significantly reduced as compared with the control, and higher ClO2 doses were more effective. A ClO2 treatment (using 3 g of generating mixture per 7.7-liter sealed container) administered 0 h after inoculation resulted in 17.6% Diplodia stem-end rot incidence compared with 95.6% in the control, whereas the same treatment administered 24 h after inoculation was much less effective, resulting in 63.0% incidence compared with 85.4% in the control. Diplodia stem-end rot incidence of naturally infected fruit after using 6 or 9 g of generating mixture per 24-liter sealed box was 23.8 or 25.7%, respectively, compared with 47.9% for control fruit. The ClO2 treatments had no negative effects on fruit quality characteristics including weight loss, firmness, puncture resistance, titratable acids (TAs), total soluble solids (TSSs), and rind color. Albedo pH at wounds was significantly reduced from 6.0 to 4.8 by the ClO2 treatments, whereas undamaged albedo remained at 5.8. In addition, no visible physiologic defects, such as peel browning and bleaching, were observed on ClO2-treated fruit. These results indicate that ClO2 gas has the potential to be developed as a component of an integrated citrus postharvest decay control system to minimize fruit losses.

Hsp70 Gene Polymorphisms Are Associated With Disease Susceptibility and HRQOL Improvement in Chinese Han Population With Systemic Lupus Erythematosus.

OBJECTIVES: The aim of this study is to investigate whether heat shock protein 70 (Hsp70) gene polymorphisms are implicated in systemic lupus erythematous (SLE) susceptibility, the efficacy of glucocorticoids (GCs) treatment, and improvement of health-related quality of life. METHODS: A total of 499 SLE patients and 499 controls were included in a case-control study, and 468 SLE patients treated with GCs for 12 weeks were involved in a follow-up study. Patients who completed the 12-week follow-up were divided into GCs-sensitive and GCs-insensitive group by using the SLE disease activity index. The SF-36 was used to evaluate the health-related quality of life of SLE patients, and genotyping was performed by improved multiplex ligation detection reaction. RESULTS: rs2075800 was associated with SLE susceptibility (adjusted odds ratio [ORadj], 1.437; 95% confidence interval [CI], 1.113-1.855; Padj = 0.005; PBH = 0.020 by dominant model; ORadj, 1.602; 95% CI, 1.072-2.395; Padj = 0.022; PBH = 0.029 by TT vs CC model; ORadj = 1.396; 95% CI = 1.067-1.826; Padj = 0.015; PBH = 0.029 by TC vs CC model). In the follow-up study, rs2075799 was associated with the improvement in mental health (p = 0.004, PBH = 0.044), but we failed to find any association between the efficacy of GCs and Hsp70 gene polymorphisms. CONCLUSIONS: Hsp70 gene polymorphisms may be associated with susceptibility to SLE and improvement of mental health in Chinese Han population.

Detection of Type, Blended Ratio, and Mixed Ratio of Pu'er Tea by Using Electronic Nose and Visible/Near Infrared Spectrometer.

The objective of this study was to find an intelligent and fast method to detect the type, blended ratio, and mixed ratio of ancient Pu'er tea, which is significant in maintaining order in the Pu'er tea industry. An electronic nose (E-nose) and a visible near infrared spectrometer (VIS/NIR spectrometer) were applied for tea sampling. Feature extraction was conducted using both the traditional method and a convolutional neural network (CNN) technique. Linear discriminant analysis (LDA) and partial least square regression (PLSR) were applied for pattern recognition. After sampling while using the traditional method, the analysis of variance (ANOVA) results showed that the mean differential value of each sensor should be selected as the optimal feature extraction method for E-nose data, and raw data comparison results showed that 19 peak/valley values and two slope values were extracted. While the format of E-nose data was in accord with the input format for CNN, the VIS/NIR spectrometer data required matrixing to meet the format requirements. The LDA and PLSR analysis results showed that CNN has superior detection ability, being able to acquire more local features than the traditional method, but it has the risk of mixing in redundant information, which can act to reduce the detection ability. Multi-source information fusion (E-nose and VIS/NIR spectrometer fusion) can collect more features from different angles to improve the detection ability, but it also contains the risk of adding redundant information, which reduces the detection ability. For practical detection, the type of Pu'er tea should be recognizable using a VIS/NIR spectrometer and the traditional feature extraction method. The blended ratio of Pu'er tea should also be identifiable by using a VIS/NIR spectrometer with traditional feature extraction. Multi-source information fusion with traditional feature extraction should be used if the accuracy requirement is extremely high; otherwise, a VIS/NIR spectrometer with traditional feature extraction is preferred.

Detecting and Monitoring the Flavor of Tomato (Solanum lycopersicum) under the Impact of Postharvest Handlings by Physicochemical Parameters and Electronic Nose.

The objective of this study was to detect and monitor the flavor of tomatoes, as impacted by different postharvest handlings, including chilling storage (CS) and blanching treatment (BT). CS tomatoes were stored in a refrigerator at 5 °C and tested at storage day 0, 3, and 7. BT tomatoes were dipped in 50 or 100 °C water for 1 min, and tested immediately. The taste, mouth feel, and aroma of tomatoes were evaluated by testing the total soluble solid content (TSS), titratable acidity (TA), ratio of TSS and TA (TSS/TA), firmness, and electronic nose (E-nose) response to tomatoes. The experimental results showed that the CS can prevent taste and firmness loss to a certain extent, but the sensory results indicated that CS accelerated flavor loss due to the TSS/TA of CS tomatoes increasing slower than control. The taste and firmness of tomatoes were impacted slightly by 50 °C BT, and were significantly impacted by 100 °C BT. Based on physicochemical parameters, different postharvest handling treatments for tomatoes could not be classified except for the 100 °C BT treated tomatoes, which were significantly impacted in terms of taste and mouth feel. The E-nose is an efficient way to detect differences in postharvest handling treatments for tomatoes, and indicated significant aroma changes for CS and BT treated tomato fruit. The classification of tomatoes after different postharvest handling treatments, based on comprehensive flavor (physicochemical parameters and E-nose combined data), is better than that based on single physicochemical parameters or E-nose, and the comprehensive flavor of 100 °C BT tomatoes changed the most. Even so, the tomato flavor change during postharvest handlings is suggested to be detected and monitored by single E-nose data. The E-nose has also been proved as a feasible way to predict the TSS and firmness of tomato fruit rather than TA or TSS/TA, during the postharvest handing process.

Novel Annonaceous acetogenins from Graviola (Annona muricata) fruits with strong anti-proliferative activity.

Five bioactive Annonaceous acetogenins, including three new compounds, annonamuricins A (1), B (2), and C (3), one registered but no spectral data reported compounds, annonamuricin D (4), and one known compound annonacin (5) were isolated from Graviola fruit (Annona muricata) and further determined through bioassay-guided fractionation. All five compounds are C35 Anonnonaceous acetogenins with a mono-tetrahydrofuran ring and four hydroxyls. Their structures were elucidated using spectral methods as well as chemical modification after isolation via chromatographic techniques and HPLC purification. These acetogenins demonstrated potent anti-proliferative activities against human prostate cancer PC-3 cells.

Effects of thermal processing and pulp filtration on physical, chemical and sensory properties of winter melon juice.

BACKGROUND: Winter melon (Benincasa hispida) is a widely consumed crop in Asia, and believed to impart special benefits to human health. The nutritional composition and sensory properties of four juice types, resulting from a combination of pulp levels (low/high pulp, LP/HP) and thermal processing (with/without boiling, B/NB), LPNB, HPNB, LPB and HPB, were compared. RESULTS: The juices had low sugars (< 20 g kg-1 ) and low titratable acidity (about 2 g kg-1 ). The insoluble solids, glucose, fructose and citric acid content in LP juice were significantly lower than in HP juice. The phenolic amino acids phenylalanine, tyrosine, and tryptophan were detected at 10-45 mg L-1 levels, and the antioxidant activity ranged from 36 to 49 mg gallic acid L-1 . C6 and C9 aldehydes were mainly found in HP juice, and boiling induced the accumulation of sulfur compounds and C5 aldehydes. The LPNB juice showed the highest acceptability in the sensory panel. The frozen concentrated orange juice (FCOJ) reconstituted with LPNB was preferable to regular FCOJ for 31% of panelists and not different for 20% of panelists. CONCLUSION: The low sugar/low acid LPNB juice with 'fresh' flavor could be developed to replace water for reconstituting FCOJ with enhanced nutritional value. © 2016 Society of Chemical Industry.

A potent antimicrobial compound isolated from Clathria cervicornis.

The sponge of Clathria cervicornis is commonly used in traditional medicine. This study aims to identify the active compound in C. cervicornis and to evaluate its antimicrobial activity. The purified active compound was determined to be crambescidin 800 and was found to be highly active against Acinetobacter baumannii (minimal inhibitory concentration, MIC=2 µg/ml), Klebsiella pneumoniae (MIC=1 µg/ml) and Pseudomonas aeruginosa (MIC=1 µg/ml). A potent antimicrobial compound, crambescidin 800, was isolated in Clathria cervicornis. It is extremely active against three common pathogenic bacteria.

Polybrominated diphenyl ethers with potent and broad spectrum antimicrobial activity from the marine sponge Dysidea.

Three polybrominated diphenyl ethers, 2-(2',4'-dibromophenoxy)-3,5-dibromophenol (1) and 2-(2',4'-dibromophenoxy)-3,4,5-tribromophenol (2) were isolated from the marine sponge Dysidea granulosa; and 2-(2',4'-dibromophenoxy)-4,6-dibromophenol (3) from Dysidea spp. They exhibited potent and broad spectrum in vitro antibacterial activity, especially against methicillin resistant Staphylococcus aureus (MRSA), methicillin sensitive Staphylococcus aureus (MSSA), Escherichia coli O157:H7, and Salmonella. Minimal inhibitory concentration (MIC) was evaluated against 12 clinical and standard strains of Gram positive and negative bacteria. The observed MIC range was 0.1-4.0mg/L against all the Gram positive bacteria and 0.1-16.0mg/L against Gram negative bacteria. 2-(2',4″-Dibromophenoxy)-3,5-dibromophenol showed stronger broad spectrum antibacterial activity than other two compounds. 2-(2',4″-Dibromophenoxy)-3,5-dibromophenol and 2-(2',4'-dibromophenoxy)-4,6-dibromophenol are thermo-stable. The results suggest that 2-(2',4'-dibromophenoxy)-3,5-dibromophenol could be used as a potential lead molecule for anti-MRSA, anti-E. coli O157:H7, and anti-Salmonella for drug development.

Three new anti-proliferative Annonaceous acetogenins with mono-tetrahydrofuran ring from graviola fruit (Annona muricata).

Bioassay-guided fractionation of the fruit powder of graviola (Annona muricata) yielded three novel compounds: muricins J, K, and L. The compounds are all C35 Annonaceous acetogenins with a mono-tetrahydrofuran ring and four hydroxyls. Their structures were elucidated by spectral methods and chemical modification after isolation via chromatographic techniques and HPLC purification. These three acetogenins demonstrated an antiproliferative against human prostate cancer PC-3 cells.

A new species of Glugea Thélohan, 1891 in the red sea bream Pagrus major (Temminck & Schlegel) (Teleostei: Sparidae) from China.

A new microsporidian species is described from farmed red sea bream Pagrus major (Temminck & Schlegel) (Teleostei: Sparidae). Large numbers of spherical whitish xenomas were observed throughout the visceral organs of the host. Histological examination showed that the microsporidia caused several xenomas that were embedded in the intestinal muscularis externa or submucosa. Light and transmission electron microscopy examination of the spores also revealed morphological features typical of species of Glugea Thélohan, 1891. This microsporidian parasite has two different types of mature spores: microspores and macrospores. The spores are elongate-ovoid, with a large posterior vacuole. The polaroplast is bi-partite, with anterior and posterior parts comprising densely packed lamellae and loose membranes, respectively, and occupies approximately the anterior half of the spore. The polar filament is anisofilar, with 12-13 coils in a single layer almost touching the posterior spore wall. Comparison of the small subunit rDNA sequences revealed 92.7-98.1% identity with the sequences available from other Glugea spp. from piscine hosts. Phylogenetic analysis demonstrated that the microsporidian species studied clustered within the Glugea clade with strong support. Based on the differences in the morphological characteristics and molecular data, the microsporidian infecting P. major is considered to represent a species new to science, Glugea pagri n. sp.

Host Status of Persian Lime (Citrus latifolia Tan.) to Oriental Fruit Fly and Mediterranean Fruit Fly (Diptera: Tephritidae) in Hawai'i.

We investigated the host status of harvest-ready green Persian lime, Citrus x latifolia Tan. (Rutaceae), to Oriental fruit fly (Bactrocera dorsalis [Hendel]) and Mediterranean fruit fly (Ceratitis capitata [Wiedemann]) (Diptera: Tephritidae) using laboratory and field studies. In forced-infestation small cage exposures (using 25 × 25 × 25 cm screened cages with 50 gravid females) and large olfactometer cage tests (using 2.9 × 2.9 × 2.5 m walk-in screened cages with 100 gravid females), punctured limes were infested by Oriental fruit fly and Mediterranean fruit fly at low rates compared to papaya controls, whereas undamaged intact fruit was not infested. Field collection and packing of 45,958 commercial export-grade fruit and subsequent incubation to look for natural infestation resulted in no emergence of fruit flies. Forced infestation studies in the field using sleeve cages to enclose fruit with a high density of fruit flies (50 gravid females) on the tree also showed no infestation. Commercial export-grade Persian lime fruit should be considered a conditional nonhost for Oriental fruit fly and Mediterranean fruit fly.

Physiochemical and Sensory Properties of a Turmeric, Ginger, and Pineapple Functional Beverage with Effects of Pulp Content.

Beverage mixtures based on pineapple juice (80-100%), with varying concentrations of turmeric (0-20%) and ginger (0-20%) juice were developed. The pineapple juice alone exhibited a total soluble solid (TSS) content of 15.90-16.03 °Brix. The total polyphenols content (TPC) varied between 0.32 and 1.79 mg GAE/mL, and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) inhibition was between 40.56% and 86.19% and correlated with the TPC and curcumin and other curcuminoids. The formulations with a high pulp content showed a significantly higher TPC and greater DPPH inhibition than those with a low pulp content. Turmeric and ginger with a high amount of pulp had a higher abundance of volatile compounds. Significant differences were observed by the panelists in the taste and mouthfeel attributes and the low-pulp juices were associated with increased palatability due to the better mouthfeel, higher sweetness, and decreased bitterness, pepperiness, pulpiness, and spiciness. The pineapple juice mixtures with 10% turmeric juice and 10% or less ginger juice were most preferred by sensory panelists.

Antifungal efficiency and mechanisms of ethyl ferulate against postharvest pathogens.

Postharvest loss caused by a range of pathogens necessitates exploring novel antifungal compounds that are safe and efficient in managing the pathogens. This study evaluated the antifungal activity of ethyl ferulate (EF) and explored its mechanisms of action against Alternaria alternata, Aspergillus niger, Botrytis cinerea, Penicillium expansum, Penicillium digitatum, Geotrichum candidum and evaluated its potential to inhibit postharvest decay. The results demonstrated that EF exerts potent antifungal activity against a wide board of postharvest pathogens. Results also revealed that its antifungal mechanism is multifaceted: EF may be involved in binding to and disturbing the integrity of the fungal plasma membrane, causing leakage of intracellular content and losing normal morphology and ultrastructure. EF also induced oxidative stress in the pathogen, causing membrane lipid peroxidation and malondialdehyde accumulation. EF inhibited the critical gene expression of the pathogen, affecting its metabolic regulation, antioxidant metabolism, and cell wall degrading enzymes. EF exhibited antifungal inhibitory activity when applied directly into peel wounds or after incorporation with chitosan coating. Due to its wide board and efficient antifungal activity, EF has the potential to provide a promising alternative to manage postharvest decay.

A novel indicator in epidemic monitoring through a case study of Ebola in West Africa (2014-2016).

The E/S (exposed/susceptible) ratio is analyzed in the SEIR model. The ratio plays a key role in understanding epidemic dynamics during the 2014-2016 Ebola outbreak in Sierra Leone and Guinea. The maximum value of the ratio occurs immediately before or after the time-dependent reproduction number (Rt) equals 1, depending on the initial susceptible population (S(0)). It is demonstrated that transmission rate curves corresponding to various incubation periods intersect at a single point referred to as the Cross Point (CP). At this point, the E/S ratio reaches an extremum, signifying a critical shift in transmission dynamics and aligning with the time when Rt approaches 1. By plotting transmission rate curves, ß(t), for any two arbitrary incubation periods and tracking their intersections, we can trace CP over time. CP serves as an indicator of epidemic status, especially when Rt is close to 1. It provides a practical means of monitoring epidemics without prior knowledge of the incubation period. Through a case study, we estimate the transmission rate and reproduction number, identifying CP and Rt = 1 while examining the E/S ratio across various values of S(0).