Tyrophagus putrescentiae (Tp) appears in contaminated foods and household surroundings. The current diagnostic tools for Tp allergy are mostly based on crude extracts and still contain shortcomings. This study aimed to investigate the immunoglobulin E (IgE)- responsiveness profiles of Tp-allergic patients and develop a molecular diagnostic method using recombinant allergens. Allergenic components were characterized as cross-reacting or species-specific allergens, in which the effective combinations of recombinant allergens were developed and analyzed in terms of the prediction accuracy for clinical diagnosis. Seven recombinant allergens were cloned and generated to detect the IgE responsiveness of the Tp allergy. A survey on the prevalence of mite allergy showed there were higher sensitizations with IgE responsiveness to house dust mites (HDM) (78.9–80.9%) than to storage mites Tp (35.6%). Prevalence of sensitization to Tp was higher in elderly subjects. The principal IgE-binding components of Tp were Tyr p 1, Tyr p 2 and Tyr p 3. Prediction accuracy for Tp allergy by IgE-responsiveness combination D  was with high precision (100%). Avoiding the cross-reactivity of Dermatophagoides pteronyssinus, the prediction accuracy of IgE-responsiveness combination H+  was suitable for Tp-specific diagnosis. Panels of Tp allergens were generated and developed a diagnostic kit able beneficial to identify IgE-mediated Tp hypersensitivity.Mite allergens are considerable factors in the genesis of allergic diseases. The storage mite  There is increasing evidence that the frequency of allergic diseases such as asthma, rhinitis and dermatitis has increased worldwide over the last few decades and becomes a global public health problem ,2,3. TheDermatophagoides pteronyssinus—are considered to be an important source for allergen sensitization and a major risk factor for allergic symptoms [D. pteronyssinus occurs at high levels of infestation in houses around the world and with high IgE frequency, which the mite extracts have indicated over 36 different allergenic components can induce IgE in patients and about 32 allergens been identified [Tyrophagus putrescentiae has been reported to cause anaphylaxis through accident ingestion of mite-contaminated foods and induce allergic respiratory symptoms after occupational or inhabited exposure [T. putrescentiae have been detected in allergic patient’s sera [D. pteronyssinus have been cloned, characterized and clarified their IgE-binding frequency or biochemical function. According to the information of World Health Organization/International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature Sub-Committee , a total of nine allergens of T. putrescentiae have been registered including Tyr p 2, Tyr p 3, Tyr p 8, Tyr p10, Tyr p 28, Tyr p 34, Tyr p 35 and Tyr p 36 [T. putrescentiae allergens is essential for the development of a diagnostic method and therapeutic agents against mite-induced allergic disorders.House dust mites—especially symptoms ,9. D. ptexposure ,11. At lTyr p 36 . The chaT. putrescentiae and house dust mite D. pteronyssinus is a frequent finding of allergic subjects in urban or humid areas [T. putrescentiae and D. pteronyssinus, which is one of the most prevalent allergens among both [T. putrescentiae and D. pteronyssinus still have not been well investigated. Evaluating the cross-reactivity between both will provide more information in which allergenic components belong to cross-reacting allergens or species-specific allergens, which is useful for allergen categorization of diagnostic agents or immunotherapeutic agents. The availability of species-specific and cross-reactive marker components creates the platform for more informative diagnostics.Cosensitization can be observed in some patients who are cosensitized independently to two or more different allergen sources in nature . The cosid areas ,15. Crosid areas ; thus, tid areas ,17. Our ong both . HoweverEscherichia coli or insect cells at low cost without risk of containing infectious materials [®) microarray-based determination of IgE against D. pteronyssinus recombinant allergens provides molecular IgE-reactivity profiles is associated with clinical manifestations in mite-allergic patients [Currently, the most commonly used standard approaches in clinical diagnosis for mite sensitization are crude extract-based methods, such as skin prick test, provocation test and serology assay ,20. Thesaterials . The avapatients .T. putrescentiae allergy have been investigated. The aim of this study was to investigate the IgE-responsiveness profiles in T. putrescentiae-allergic patients. Evaluating the cross-reactivity of these allergenic components between T. putrescentiae and D. pteronyssinus, to characterize allergens belong to cross-reactive allergens or species-specific allergens, which we can develop the effective agent combination for clinical diagnosis.To date, no relevant studies focusing on the development of differential diagnostic kit for D. pteronyssinus, D. farinae and T. putrescentiae.A total of 298 allergic subjects  who attended the Clinic in the Division of Allergy, Immunology and Rheumatology at Taichung Veterans General Hospital (TCVGH) were recruited for this study. These allergic patients were diagnosed with allergies by a specialist attending physician if they had at least one of the following history: asthma, rhinitis, atopic dermatitis or eczema. The collection of blood samples from patients was approved by the Institutional Review Board, TCVGH (TCVGH IRB No. C07126). Written informed consent was obtained from each participant before being enrolled in the study. Blood samples were drawn, and sera were stored for the measurement of specific IgE to D. pteronyssinus were prepared from lyophilized whole mite bodies purchased from Allergon . The T. putrescentiae mites were cultured in our laboratory with a medium consisting of yeast extract and mouse chow, then mite bodies were separated from the medium. The mite collection is described in our previous study [T. putrescentiae mites were homogenized and extracted with phosphate buffer saline . Protein concentration was determined by Bradford assay  using bovine albumin as a standard.Two kinds of mite crude extracts were used in this study. The crude extracts of us study . In brie3, pH 8.4 for 4 h at RT. After blocking with 1% skimmed milk, it was then incubated for 1 h at room temperature [2SO4 after 15 min, and the optical density was measured at 450 nm in a multiscan spectrophotometer . The cutoff value was mean ± 2x standard deviation as 0.23 ± 0.02 unit of the sera of 10 healthy volunteers who have no history of allergic symptoms, and samples exceeding 0.25 unit were considered as positive.The IgE responsiveness against mite crude extracts in sera of allergic patients was determined by an enzyme-linked immunosorbent assay (ELISA) as previously described . Brieflyperature . Wells wT. putrescentiae were prepared and used to analyze the frequency of IgE reactivity by sera from the allergic subjects. Total RNA of mites was isolated with Trizol reagent, and cDNA was synthesized with primer dT, MMLV reverse transcriptase . The recombinant proteins of T. putrescentiae allergenic components were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) from cDNA and subcloned into the expression vector. The specific primers were designed based on the accession number in the National Center for Biotechnology Information or in our previous study finding. The program of PCR was carried out at 94 °C for 10 s, 55 °C for 20 s and 72 °C for 3 min for 35 cycles. The PCR products were purified, ligated into a pQE30 vector and transformed in Escherichia coil-M15 strain competent cells. Transformants were selected by kanamycin (25 µg/mL) and ampicillin (100 µg/mL). Expressions of recombinant allergens were performed according to the methods described in the QIA ExpressionistTM Kit directions . The recombinant allergens were expressed as a 6x His-tagged protein using 1-mM isopropyl-β-D-thiogalactopyranoside  induction. The proteins were purified by nickel-nitrilotriacetic acid agarose metal affinity column chromatography under native conditions.The recombinant allergens  of D. pteronyssinus then to evaluate the IgE responsiveness for identification the species-specific components of T. putrescentiae. For the IgE absorption, serum samples were selected from 106 allergic subjects who had IgE antibodies against T. putrescentiae crude extracts using ELISA. Each serum was preincubated with 50 µL of D. pteronyssinus crude extracts (50 µg/mL) and left overnight at 4 °C. After the preabsorption, the subsequent procedures for determining IgE responsiveness to T. putrescentiae by ELISA were the same as those described previously.Because of the cross-reactivity between different mite species, the patients’ sera were pre-absorbed with T. putrescentiae were prepared and used to investigate the frequency of IgE responsiveness. The IgE antibodies in serum samples were detected by ELISA. The recombinant allergens  were coated separately onto wells of polyvinyl microtiter plates  by the addition of 100 μL of a 5-μg/mL solution in 0.1 MNaHCO3 (pH 8.4) for 4 h at RT. After blocking with 1% skimmed milk, it was then incubated for 1 h at RT. Wells were washed with PBS containing 0.05% Tween-20 (PBST). The ratio 1:5 dilution of serum from each allergic patient was added in 100 µL PBS. Wells were washed again with PBST, peroxidase-conjugated anti-human IgE (1:1000 dilute in PBST) was added to the wells and incubated for 2 h at RT. After washing with PBST, the bound enzyme substrates were detected with TMB substrate . The reaction was stopped with 50 μL 1-N H2SO4 after 15 min, and the optical density was measured at 450 nm in a multiscan spectrophotometer . The cutoff value was mean ± 2x standard deviation as 0.23 ± 0.02 unit of the sera of 10 healthy volunteers who have no history of allergic symptoms, and samples exceeding 0.25 unit were considered as positive. The IgE-binding frequency of T. putrescentiae allergens among allergy patients was analyzed and compared after D. pteronyssinus absorption. Each serum was pre-incubated with 50 µL of D. pteronyssinus crude extracts (50 µg/mL) and left overnight at 4 °C. After the pre-absorption, the subsequent procedures for determining IgE responsiveness to T. putrescentiae allergens by ELISA were the same as those described previously.Recombinant allergens of T. putrescentiae were cloned and the recombinant allergens were generated for detection combinations of IgE responsiveness in sera from allergic patients. The higher frequency with IgE responsiveness of allergenic components are picked out, the higher sensitivity of detection combination for T. putrescentiae can be obtained. Since the cross-reactivity of T. putrescentiae and D. pteronyssinus were common in clinical practice. Sera from allergic patients with the specificity of T. putrescentiae allergy were confirmed after the absorption of D. pteronyssinus. Only those detection combinations with both high sensitivity and specificity will be used for clinical application in the future.A total of seven allergenic components of T. putrescentiae allergy were analyzed by the nonparametric alternative to the unpaired two-sample t-test. The p-values of differences less than 0.05 were considered statistically significant.Differences between the middle-age and elderly age population of the D. pteronyssinus, D. farinae and T. putrescentiae of allergic patients was determined by ELISA with mite crude extracts. The results showed that the IgE reaction with positive responses of allergic subjects were 80.9% (241/298) to D. pteronyssinus, 78.9% (235/298) to D. farinae and 35.6% (106/298) to T. putrescentiae  and storage mite T. putrescentiae were 33.6% (100/298)  (T. putrescentiae (35.6%).The sensitization to shown in A. There 100/298) B. The pr134/298) B. There D. pteronyssinus and T. putrescentiae were compared between elderly and young adult subjects. The results showed there were 84.1% (116/138) allergic subject sensitive to D. pteronyssinus and 81.2% (112/138) sensitive to D. farinae in the young adult group; there was a slightly higher prevalence of D. pteronyssinus sensitivity in the young adult group than in the elderly group (T. putrescentiae while there were only 25.4% (35/138) young adult subjects allergic to T. putrescentiae. The prevalence of sensitization to D. pteronyssinus or D. farinae was slightly higher in the young adult subjects, while the prevalence of sensitization to T. putrescentiae was significantly higher in elderly subjects than in young adult subjects (44.4% vs. 25.4%) (p < 0.05).Differences in sensitization to ly group . AdditioT. putrescentiae was isolated from alive mite bodies and the complementary DNA (cDNA) synthesized via reverse transcription [T. putrescentiae allergens was amplified by RT–PCR and subcloned into expression vectors. A total of seven allergenic components were expressed and purified by His-tag affinity column, including rTyr p 1, rTyr p 2, rTyr p 3, rTyr p 7, rTyr p 8, rTyr p 10 and rTyr p 20. The characteristics including biologic function, molecular weight and accession No. of allergens from T. putrescentiae we used in this study is listed in T. putrescentiae recombinant allergens on SDS-PAGE showed that the rTyr p 2 approximately at 16 kDa, the rTyr p 1, rTyr p 3, rTyr p 7 and rTyr p 8 approximately at 24–26 kDa and rTyr p 10 & rTyr p 20 approximately at 37–40 kDa , Tyr p 2 (79.2%), Tyr p 3 (49.1%), Tyr p 7 (37.7%), Tyr p 8 (44.3%), Tyr p 10 (18.9%) and Tyr p 20 (48.1%), respectively (T. putrescentiae were Group 1 (Tyr p 1) and Group 2 (Tyr p 2) with a molecular weight of 25 kDa (61.3%) and 16 kDa (79.2%). The Group 3 allergen (Tyr p 3) with a molecular weight of 26 kDa (49.1%) was also one of the most prevalent IgE-binding components. The IgE-binding components of Group 8 (Tyr p 8) with a molecular weight of 26 kDa (44.3%) and Group 20 (Tyr p 20) with a molecular weight of 40 kDa (48.1%) also belonged to significant allergens. For further confirmation of cross-reactivity between D. pteronyssinus, the T. putrescentiae-sensitive sera were selected for performing the IgE immunoblot inhibition with D. pteronyssinus absorption. After D. pteronyssinus absorption, the IgE-binding frequency of Tyr p 1, Tyr p 2, Tyr p 3, Tyr p 7, Tyr p 8, Tyr p 10 and Tyr p 20 were decreased as 51.9%, 39.6%, 41.5%, 32.1%, 17.9%, 5.7% and 17.9% , following by Tyr p 2, (two-fold decreased). The level of cross-reactivity between T. putrescentiae and D. pteronyssinus was trivial in Tyr p 1, Tyr p 3 and Tyr p 7. Results showed the highest level of cross-reactivity between T. putrescentiae and D. pteronyssinus were Group 10 allergens, the following were Group 20 and Group 8 allergens. The protein sequences of these allergens were compared between T. putrescentiae and D. pteronyssinus by Basic Local Alignment Search Tool (BLAST), it showed the sequence similarities were over 80% in Group 8, Group 10 and Group 20 allergens  in the elderly age group  and 25.4% (35/138) in the middle-age group (less 40 years old). For further identification of the allergy condition in different age groups, the sera with positive IgE responsiveness to T. putrescentiae were selected for the measurement of responsiveness to each T. putrescentiae allergenic components before and after D. pteronyssinus absorption. Before the D. pteronyssinus absorption, the frequencies of IgE-binding to each T. putrescentiae allergenic components were shown as follows: Tyr p 1 (63.6%), Tyr p 2 (78.9%), Tyr p 3 (43.7%), Tyr p 7 (39.4%), Tyr p 8 (43.7%), Tyr p 10 (12.7%) and Tyr p 20 (47.9%) in the elderly age group, respectively , Tyr p 2 (80%) and Tyr p 3 (60%). After the D. pteronyssinus absorption, the frequencies of IgE-binding to Tyr p 1 (57.7% vs. 40%), Tyr p 2 (49.3% vs. 20%), Tyr p 7 (35.2% vs. 25.7%) and Tyr p 8 (19.7% vs. 14.3%) in the elderly age group were higher than in middle-age group. The data of statistical analysis showed the frequencies of IgE-binding were significantly reduced after the D. pteronyssinus absorption in the Group 10 and Group 8 allergens no matter in the elderly age group or middle-age group , combination B (Tyr p 2 & Tyr p 3), combination C (Tyr p 3 & Tyr p 7), combination D  and combination E  to determine the prediction accuracy. All possible permutations of allergen IgE responsiveness were analyzed to find out the most accurate combination set. Because of the size of the table, only a few combinations of five sets with high predictive value over 80% of T. putrescentiae allergy are shown in T. putrescentiae allergy by IgE-responsiveness combination A, B and C could be reached up to eighty percent (84.0–88.7)  and E  could be achieved to 100% (The identification of sensitization to storage mite .0–88.7) . The pre to 100% . It seemT. putrescentiae and D. pteronyssinus was used to evaluate the cross-reactivity and to identify different components belongs to cross-reacting allergens or species-specific allergens. In order to consider the influence of cross-reactivity from house dust mite, the IgE adsorption experiment was performed. The data of IgE-responsiveness combinations to species-specific allergens with high predictive value over 55% of T. putrescentiae allergy are shown in D. pteronyssinus absorption, the IgE responsiveness to T. putrescentiae allergenic components were chosen to composite seven combinations, including combination A+ (Tyr p 1 & Tyr p 2), combination B+ (Tyr p 2 & Tyr p 3), combination C+ (Tyr p 3 & Tyr p 7), combination D+ , combination E+ , combination F+  and combination G+ . The prediction accuracy for T. putrescentiae allergy by IgE-responsiveness combination D+  could be reached up to 90%  of storage mite sensitization [T. putrescentiae, which is much higher than D. pteronyssinus (22%) in the same age group. Similar findings have been reported in Korea. T. putrescentiae is the third most common mite in the indoor environment and around one-third of individuals are allergic to it [T. putrescentiae sensitization in the general adult population, especially in individuals over 50 years old, there are few reports on its clinical significance in elderly populations. It is of interest to evaluate the allergenicity, prevalence of hypersensitivity and clinical relevance of T. putrescentiae sensitization in elderly populations, this phenomenon implicates that D. pteronyssinus and T. putrescentiae may have different sensitization exposure.Inhaled allergens are considerable factors in the genesis of allergic diseases. The ubiquitous presence of oundings . In viewdiseases ,31. The n Taiwan , and the regions . The phemponents . There ih Europe  and Asiah Europe . The prenyssinus . The sto America . T. putrf Africa . In Spaiic to it . DespiteB. tropicalis and house dust mite D. pteronyssinus by skin tests and the molecular structure and cross-reactivity between Blo t 5 and Der p 5 were estimated at 33% to 43% by IgE inhibition [D. pteronyssinus, but the others are species-specific allergens [T. putrescentiae and D. pteronyssinus allergy in allergic rhinitis individuals by using skin prick tests, basophil histamine release assays in vitro and specific IgE measurements [T. putrescentiae and D. pteronyssinus was investigated and results showed that the major cross-reacting allergen was Group 2 allergens and species-specific allergen was Group 3 allergens [Recently, storage mites have been reported to be clinically important allergenic components of dust samples throughout the world, supporting their clinical significance . In our hibition . This sullergens . In our urements . The reallergens .T. putrescentiae to cause immune responses for developing allergic diseases. For allergy diagnosis, the purity and the representativeness of the allergens should also be considered. Since the diagnosis of T. putrescentiae allergy is still based on the crude extracts and some of the allergenic components which been found or obtained. Some of these allergenic components possess enzymatic activities causing the instability of allergen preparation which results in IgE-binding activity varied and difficult to make an accurate diagnosis. That is the explanation why the divergences of allergen contents had been mentioned that the levels of both Der p 1 and Der p 2 in D. pteronyssinus extract from the different company reveal variability and the proportion variation of Der p 1 and Der p 2 among different extracts are likely to influence their biologic effectiveness [© ISAC allergen-microarray  for the determination of specific IgE against recombinant or purified allergen components has good analytical performance when compared with the traditional method of ImmunoCAP© 250 [T. putrescentiae sensitized patients. The prediction accuracy for T. putrescentiae allergy by IgE-responsiveness combination D  and E  could be achieved to 100%. It seems the prediction combinations of these recombinant allergens from major allergens are suitable for diagnostic agents. Recombinant allergens have provided us with high accuracy and efficiency that can improve the diagnosis of allergy.It is also a paramount challenge to develop a small amount of blood for the diagnosis of mite allergy. The study can provide a solution to detect many allergens with a small amount of blood to identify which allergenic components of tiveness . Therefotiveness . The CRDtiveness . The pottiveness . CRD enativeness . CRD cantiveness . The ImmCAP© 250 . In our Childhood asthma and adult-onset asthma are a globally significant immune-disorder disease with major public health consequences known to share many of the same causes and triggers . AllergiT. putrescentiae allergenic components and develop an immunoassay kit to identify the IgE-mediated hypersensitivity of T. putrescentiae. A patent for the diagnostic kit (patent No. I 467177) for T. putrescentiae allergy including the allergens of rTyr p2, rTyr p3, rTyr p 7, rTyr p 8, rTyr p 10 and rTyr p 20 has been registered and certified in Taiwan, the Republic of China (expired time 04/22/2032). This is based on a combination with high-frequency IgE-binding component of Tyr p 2 and the high species-specific allergenic components of Tyr p 3 & Tyr p 7, a refined microarray panel of allergens. The development of diagnostic kits can be identified T. putrescentiae IgE-mediated hypersensitivity more specifically the detectable allergens would broaden involved more species-specific allergens in T. putrescentiae, which avoid the cross-reactive allergen of D. pteronyssinus whenever possible.It is feasible to generate a panel of Tyrophagus putrescentiae is one of the most common storage mites found in the storage rooms, kitchen and frequently contaminated in the mushrooms, farina and pet food [D. pteronyssinus and D. farinae) and storage mites (T. putrescentiae) be identified in the dust samples [T. putrescentiae is the most prevalent mite in all room types [T. putrescentiae can be accumulated in the contaminated foods or residential surroundings causing food allergy and airway hypersensitivity [D. pteronyssinus, in contrast only a few allergens of T. putrescentiae been identified and characterized. In the currently identified allergens of T. putrescentiae, there are some major allergens such as Tyr p 1, Tyr p 2 and Tyr p 3 with higher than 50% IgE-binding activity in the T. putrescentiae sensitized patients caused hypersensitivities.pet food . There a samples ,49. Antisitivity ,50. AlthD. pteronyssinus absorption indicating certain levels of cross-reactivity between T. putrescentiae and D. pteronyssinus. In this study, the highest level of cross-reactivity allergen between T. putrescentiae and D. pteronyssinus was the Group 10 mite allergen. The Group 10 mite allergens are involved in muscle contraction in invertebrates and processed high levels of cross-reactivity between mites, shrimp and insects in seafood-allergic patients, referred to as pan-allergens [T. putrescentiae and D. pteronyssinus was confirmed after the IgE absorption and protein sequence comparison; it showed the most cross-reactive allergens were Group 10, Group 20 and Group 8. Another highly conserved component had been identified as Group 2 allergen, similar findings demonstrated that the IgE-binding activity of Tyr p 2 could be extremely absorbed by Der p 2 and the Group 2 allergens are the major cross-reactive allergen of T. putrescentiae and D. pteronyssinus [T. putrescentiae-allergic patients. Most the surface residues are conserved leading to similar epitopes forming the structural basis of cross-reactivity between Group 2 allergens of dust mites [T. putrescentiae and D. pteronyssinus, such as Tyr p 3 and Tyr p 7, the reduction of IgE-binding activity after D. pteronyssinus absorption were trivial. The similar finding showed there are poor correlations of serum specific IgE to Tyr p 3 and Der p 3, which with poor cross-reactivity between them and the inhibition degree by Group 3 allergens (Tyr p 3) is lower than by Group 2 allergens (Tyr p 2) in the T. putrescentiae-allergic patients [T. putrescentiae specific allergen and may serve as a suitable candidate for a differential diagnostic marker for T. putrescentiae hypersensitivity.The seven allergenic components were used to identify the IgE-binding activity and to confirm the IgE immunoblot inhibition, it showed that all of the IgE-binding activities decreased after llergens . The cronyssinus . The Dernyssinus , which bnyssinus . The allnyssinus , therefost mites . Not allpatients . OverallT. putrescentiae-allergic patients. Not all of the identified allergens of T. putrescentiae were used for the development of diagnostic kits in this study. Although not all allergenic components are included, most major allergens are included for diagnosis and the prediction accuracy is close to 100%. In this study, just 106 T. putrescentiae-allergic patients were used to check the accuracy of diagnostic kits. Actually, a total of 298 allergic patients were recruited in the Clinic of the Division of Allergy, only about 1/3 of allergic patients were allergic to T. putrescentiae with IgE positive responses. This is can be contributed to more than 80% of the dust samples contained house dust mites including D. pteronyssinus and D. farinae in Taiwan [D. pteronyssinus and D. farinae [D. pteronyssinus and D. farinae, the major predominant mites that elicit positive IgE responses and provoke airway hypersensitivity in Taiwan [The main limitations of this study are related to the usage of allergen groups and the recruitment of n Taiwan . Most of farinae . Becausen Taiwan . Anothern Taiwan . In thisT. putrescentiae is an important occupational hazard in agricultural environments, accident ingestion of mite-contaminated foods and ubiquitous presence in urban or rural settings, it is also a common allergy-provocation factor in household surroundings. So, the diagnosis method of T. putrescentiae allergy with convenience and accuracy is necessary for clinical treatment. The allergy diagnosis based on the crude extracts still contains some disadvantages and insufficiencies. The recombinant allergens can be used for understanding the sensitization profiles of allergic patients. In this study, we found out prediction accuracy of IgE responsiveness for T. putrescentiae with allergen combination  could be achieved to high precision (100%). Avoiding the allergen cross-reactivity from predominant mite D. pteronyssinus performed by IgE absorption, the allergenic components of Tyr p 3 and Tyr p 7 have been identified as species-specific allergens. The IgE responsiveness to species-specific allergens is more suitable for the diagnosis of T. putrescentiae allergy focusing on IgE-mediated hypersensitivity.The storage mite


In the article titled “Inhibition of cAMP/PKA Pathway Protects Optic Nerve Head Astrocytes against Oxidative Stress by Akt/Bax Phosphorylation-Mediated Mfn1/2 Oligomerization”  and Akt regulates Bax phosphorylation at Ser184, inhibiting Bax effects on the mitochondria [40], we further determined the effect of PKA inhibition on Akt and Bax phosphorylation in ONH astrocytes against oxidative stress combined with cAMP elevation. We observed that PKA inhibition promoted cell viability/mitochondrial activity in ONH astrocytes against oxidative stress combined with cAMP elevation ). Our results demonstrated that PKA inhibition significantly promoted the phosphorylation of Akt at Ser473 and Bax at Ser184 in ONH astrocytes against oxidative stress combined with cAMP elevation ). Collectively, our findings suggest that inhibition of the cAMP/PKA pathway promotes the survival of ONH astrocytes against oxidative stress combined with cAMP elevation by Akt/Bax phosphorylation-mediated Mfn1 and Mfn2 oligomerization .


Whole genome sequencing is effective at identification of small variants, but because it is based on short reads, assessment of structural variants (SVs) is limited. The advent of Optical Genome Mapping (OGM), which utilizes long fluorescently labeled DNA molecules for de novo genome assembly and SV calling, has allowed for increased sensitivity and specificity in SV detection. However, compared to small variant annotation tools, OGM-based SV annotation software has seen little development, and currently available SV annotation tools do not provide sufficient information for determination of variant pathogenicity.nanotatoR, which provides comprehensive annotation as a tool for SV classification. nanotatoR uses both external  and internal (user-defined) databases to estimate SV frequency. Human genome reference GRCh37/38-based BED files are used to annotate SVs with overlapping, upstream, and downstream genes. Overlap percentages and distances for nearest genes are calculated and can be used for filtration. A primary gene list is extracted from public databases based on the patient’s phenotype and used to filter genes overlapping SVs, providing the analyst with an easy way to prioritize variants. If available, expression of overlapping or nearby genes of interest is extracted . Most quality-control filtration parameters are customizable by the user. The output is given in an Excel file format, subdivided into multiple sheets based on SV type and inheritance pattern .We developed an R-based package, nanotatoR passed all quality and run time criteria of Bioconductor, where it was accepted in the April 2019 release. We evaluated nanotatoR’s annotation capabilities using publicly available reference datasets: the singleton sample NA12878, mapped with two types of enzyme labeling, and the NA24143 trio. nanotatoR was also able to accurately filter the known pathogenic variants in a cohort of patients with Duchenne Muscular Dystrophy for which we had previously demonstrated the diagnostic ability of OGM.The extensive annotation enables users to rapidly identify potential pathogenic SVs, a critical step toward use of OGM in the clinical setting. With the advent of the high-throughput short-read sequencing (SRS) techniques, identification of molecular underpinnings of genetic disorders has become faster, more accurate and cost-effective . SRS plaChromosomal microarray (CMA) is the established method for high-accuracy detection of CNVs, but it can only identify gains or losses of genetic material and is virtually blind towards identification of balanced rearrangements such as inversions or translocations. CMA clinical application is typically limited to CNVs above 25–50 kb, although higher resolution CNV maps have been built and are being used to design disease-specific paths to diagnostic detection of smaller variants (e.g. ). BreakpNovel approaches which analyze single, long DNA molecules hold the promise of detecting the previously inaccessible SVs. Long-read sequencing (LRS) technologies such as nanopore-based sequencing (Oxford Nanopore Technologies) or single-molecule real-time sequencing (Pacific Biosciences) have the potential to both detect complex genomic rearrangements and increase SV break point resolution , 14. Thede novo genome assembly. Assembly and variant calling are performed using algorithms provided by Bionano Genomics and/or tools developed by the community, such as OMblast  in (b). This variant deletes the entirety of coding gene UGT2B17, as well as the 3 pseudogenes UGT2B29P, AC147055.2 (or RP11-1267H10.2) and AC147055.4 (or RP11-1267H10.1), and ~ 63% of pseudogene AC147055.3. c) nanotatoR snapshot: The nanotatoR output indicates the overlap genes, the strand from which they are transcribed, and the percentage of the gene length overlapping with the deletion. It also displays the frequency (expressed as percentage) of the variant in the internal and BNDB databases, and the overlapping gene term with database info (osteoporosis found in the OMIM database).Additional file 2: Supplementary Table S1. NA24385 nanotatoR-annotated structural variant results. Content of the various tabs is detailed in the first tab (“LegendS1”) of the Excel workbook. SVs reported in the indel, inv., and trans tabs have undergone the default nanotatoR filtration . Columns in each sheet of the workbook are either a direct output of SVcaller or appended by nanotatoR, as indicated in column 3 of the table. Details about SVcaller columns are available at https://bionanogenomics.com/wp-content/uploads/2017/03/30041-SMAP-File-Format-Specification-Sheet.pdf.Additional file 3: Supplementary Table S2. Primary gene list generated for NA24385 (GM24385) dataset filtration using the term Osteoporosis. A total of 307 genes were extracted (the UGT2B17 gene is highlighted). Columns show: Genes: Primary gene symbols. Terms: Query term associated with each gene. Each cell shows the gene symbol and, between parentheses, the query term and the database  from which each gene was extracted. Clinical Significance: Only genes for which likely pathogenic or pathogenic variants were found in ClinVar are displayed in this column. The parentheses match the database order in column 2, displayed as  to indicate the information is from ClinVar and not Gene or OMIM.Additional file 4: Supplementary Table S3. Calculation of internal frequency for the deletion variant overlapping UGT2B17 in sample NA24385. Content of the tabs and column names is detailed in the first tab (“LegendS3”) of the Excel workbook. The columns used to calculate the filtered and unfiltered frequencies are highlighted in the del_totalData tab.Additional file 5: Supplementary Table S4. Calculation of BNDB database frequency for the deletion variant overlapping UGT2B17 in sample NA24385. Content of the tabs and column names is detailed in the first tab (“LegendS4”) of the Excel workbook. The columns used to calculate the filtered and unfiltered frequencies are highlighted in the GM24385_data_all tab.Additional file 6: Supplementary Table S5. Typical run times in minutes for the various tasks for the control sample datasets. nanotatoR functions are indicated in italics in Column 1. Processing times for the Ashkenazi trio DLE labeling dataset (Example I) are shown in Column 1 (no RNA-Seq data was available for this sample). Processing times for the singleton sample (Example II) DLE labeling and dual enzyme labeling (SVmerge) are shown in columns 2 and 3 respectively.Additional file 7: Supplementary Table S6.nanotatoR annotation of structural variants in the DLE-labeled sample NA12878 dataset. Content of the various tabs is detailed in the first tab (“LegendS6”) of the Excel book. SVs reported in the indel, inv., and trans tabs have undergone the default nanotatoR filtration . Columns in each sheet of the workbook are either a direct output of SVcaller or appended by nanotatoR, as indicated in column 3 of the table. In the all_PG_OV tab, the cells containing the query genes are highlighted in purple. Details about SVcaller columns are available at https://bionanogenomics.com/wp-content/uploads/2017/03/30041-SMAP-File-Format-Specification-Sheet.pdfAdditional file 8: Supplementary Table S7.nanotatoR annotation of structural variants in the dual-labeled sample NA12878 dataset (SVmerge output). Content of the various tabs is detailed in the first tab (“LegendS7”) of the Excel book. SVs reported in the indel, inv., and trans tabs have undergone the default nanotatoR filtration . Columns in each sheet of the workbook are either a direct output of SVcaller or appended by nanotatoR, as indicated in column 3 of the table. In the all_PG_OV tab, the cells containing the query genes are highlighted in purple. Details about SVcaller columns are available at https://bionanogenomics.com/wp-content/uploads/2017/03/30041-SMAP-File-Format-Specification-Sheet.pdfAdditional file 9: Supplementary Table S8. Genes overlapping deletion variants for NA12878. Chromosome and SV breakpoint start and end are shown in: - NA12878  hg38 annotation): Overlapping genes coordinates identified in the original paper by Mak et al. 2016, with hg38 reference genome annotation. - NA12878  hg19 liftOver): Overlapping genes coordinates identified in the original paper by Mak et al. 2016, lifted over to hg19. - NA12878 (nanotatoR SVmerge annotation): Overlapping genes coordinates for NA12878 labelled by BSPQI and BSSSI enzyme (SVmerge), as annotated by nanotatoR. - NA12878 (nanotatoR DLE annotation): Overlapping genes coordinates for NA12878 labelled by DLE1 enzyme (SVmerge), as annotated by nanotatoR.Additional file 10: Supplementary Table S9. Variant annotation for DMD samples. Singleton samples CDMD_1003 and CDMD_1159 were analyzed with single DLE labeling and are shown in the SingleLabel_Solo tab. Singleton samples CDMD_1155, CDMD_1156, and CDMD_1187 were analyzed with dual enzyme labeling and are shown in the DualLabel_Solo tab. Mother proband dyads CDMD_1131, CDMD_1157, and CDMD_1163 were analyzed with dual enzyme labeling and are shown in the DualLabelDuo tab. The columns used for Table nanotatoR, as indicated in column 3 of the table in the first tab (“Legend S9”). Details about SVcaller columns are available at https://bionanogenomics.com/wp-content/uploads/2017/03/30041-SMAP-File-Format-Specification-Sheet.pdf.


The inhibition is strongly dependent on concentration of dodecyl-maltoside (DM) in the assay. The plots of Ki vs [DM] are linear for both estradiol and testosterone which may indicate an 1:1 stoichiometry competition between the hormones and the detergent. Binding of estradiol, but not of testosterone, brings about spectral shift of the oxidized CcO consistent with an effect on heme 3a3+. We presume that the hormones bind to CcO at the bile acid binding site described by Ferguson-Miller and collaborators. Estradiol is shown to inhibit intraprotein electron transfer between hemes a and 3a. Notably, neither estradiol nor testosterone suppresses the peroxidase activity of CcO. Such a specific mode of action indicates that inhibition of CcO activity by the hormones is associated with impairing proton transfer via the K-proton channel.Estradiol, testosterone and other steroid hormones inhibit cytochrome  Iteviewed, ) and gaseviewed, ) has bee example ,9,10,11)c oxidase (CcO) from Rhodobacter sphaeroides or bovine heart mitochondria. The site is located near the inner side of the membrane close to the entrance of the so-called K-proton channel and binds a variety of amphiphilic ligands of diverse nature sharing structural similarity, such as bile acids, thyroid hormones, retinoic acid and many other compounds; see Buhrow et al.  is shown in i value of 0.37 mM. The estimated dissociation constant for the competitor, DM, in the absence of estradiol is Kc = 1.47 mM.The plot of K1) the inhibition directly observed can be as high as 3-fold and occurs instantaneously. As with estradiol, the inhibitory effect of testosterone depends on DM concentration, but in a more complex way. Addition of excess testosterone (4 mM) at low concentration of DM (1 mM) inhibits the activity but weakly, whereas strong inhibition is induced by this concentration of the hormone in the presence of 20 mM DM . Accordingly, when DM concentration is raised in the course of respiration (trace 3), the inhibition by the initially present excess testosterone (3 mM) is not released as it was observed with estradiol but rather is slightly augmented.Inhibition of cytochrome oxidase activity by testosterone is shown in 2–5) and as with estradiol, we assign this lag phase to hormone binding with the empty DM micelles.The complex pattern of CcO inhibition by testosterone can be better understood from the titrations made at different concentrations of DM B. At lowi(app), the titration curves in v, with testosterone concentration, [I]. It is similar to Equation (1) but has an additional parameter R which denotes the fraction of the activity resistant to inhibition:In order to determine Ki(app), L and R) were found to depend on DM concentration. In particular, the dependence of the lag phase on [DM] is linear throughout the entire concentration range explored, i.e., 1–40 mM (see the Inset in Equation (2) fits well the experimental data after the end of the lag-phase (see other details in the legend). All the parameters (Ki(app) on [DM] is shown in i:Kc = 0.05. Extrapolation to zero DM concentration yields the true Ki value for testosterone as low as 80 µM, whereas the Kc value is now determined as 1.3 mM (compare to 1.47 mM as estimated in The dependence of K1–4) but much better spectra are obtained at 1 mM of the hormone (spectra 5–7 in i(app) value obtained in the activity inhibition studies  with a maximum at 437 nm and a trough at 415 nm is typical of a red shift of ferric heme 3a Soret band induced by strong ligands such as cyanide. The magnitude of 10 mM−1⋅cm−1 obtained at 1 mM of the hormone corresponds to ca. 1/5 of the effect induced by cyanide, see van Buuren et al. [2) in reasonable agreement with the time course of the inhibitory action of estradiol that takes a few minutes to develop .The spectral perturbation of CcO induced by estradiol is strongly modified by cyanide B. The S-3a. Perturbation of the heme 3a spectrum would be consistent with the proposal by Hiser et al. [These data allow to suggest that the spectral perturbation of the oxidized ligand-free CcO induced by estradiol is associated with heme r et al.  that theTo the best of our knowledge, mechanism of the inhibitory action of the BABS ligands on CcO activity has not been studied experimentally so far.a to heme 3a. To this end, the kinetics of the hemes’ reduction by dithionite in the presence of RuAm was studied. In the experiment shown in a is almost fully reduced within the mixing time as evidenced by the spectra in the α-band region , which is followed by time-resolved electron transfer to heme 3a observed in the Soret region  [We investigated effect of estradiol on the kinetics of electron transfer from heme , Soret) ,21.3a reduction monitored at 445 nm vs the 407 nm reference is noticeably decelerated in the presence of 200 µM and, especially, 500 µM estradiol . The effect was reproduced with several samples.As shown in 3a. In the control (trace 1), the rate constants are 105.3 s−1 and 6.15 s−1, which is in agreement with our earlier data at these concentrations of dithionite and RuAm [2) the rate constants change to 67.75 s−1 and 5.3 s−1. An increase in the estradiol concentration to 0.5 mM (trace 3) yields a further decrease of the values to 39.6 s−1 and 2.5 s−1, respectively. The contributions of the rapid and slow phases make up about 70% vs 30% in all three cases. These estimates are consistent with the data obtained in the experiments with an oxygen electrode. For example, according to the rapid mixing data, 0.5 mM estradiol decreases by ca. 60% the rate constants of the both phases (without a notable effect on their contributions), and the same concentration of estradiol inhibits oxygen consumption approximately by half  is two oxidizing equivalents deficient relative to the resting oxidized state of CcO and is homologous to Compound I of peroxidases. The second oxygen atom is subsequently reduced to H2O by two sequential electron transfers from exogenous substrate, a process fully analogous to reduction of Compound I to the resting state in peroxidases. Formation of this second water molecule by CcO was denoted therefore as the peroxidase phase of the CcO catalytic cycle. The protons consumed in H2O formation in the peroxidase phase are delivered exclusively via the D-proton channel and the K-channel is not involved in this part of the CcO catalytic cycle.Taking into consideration close proximity of the BABS to the entry point of the K-proton channel, it was speculated that binding of the ligands to the amphipathic site could result in impairment of proton transfer to the binuclear site via the K-pathway, see Hiser et al. . Reductitantinov ), O2 bou2O2 instead of O2 as the electron acceptor [R. sphaeroides do not affect the peroxidase activity of CcO while fully inhibiting the oxidase activity of the enzyme, see Vygodina et al. [The binuclear site of CcO can use Hacceptor ,22. In ta et al. . TherefoOur work shows that testosterone and estradiol can inhibit significantly isolated cytochrome oxidase from bovine heart mitochondria. The inhibition of the isolated bovine CcO has been observed also with a number of other steroid hormones including progesterone and DHEA, as well as with vitamin D which is classified as secosteroid .−5–10−4 M concentration. All of them revealed competition with dodecyl-maltoside. None of the assayed compounds inhibited peroxidase activity of CcO, with the exception for cholecalciferol which, however, affects peroxidase reaction in concentrations an order of magnitude higher than oxidase activity , while R. sphaeroides whereas bovine heart oxidase was studied in our experiments. It would be tempting to speculate that the animal CcO can be more susceptible to inhibition by animal hormones than the bacterial enzyme, emphasizing physiological relevance of the effect. However, discrepancy between the data here and in [1). In order to clarify the issue, it would be best to carry out side-by-side experiments with the bovine and bacterial oxidases under identical conditions. Our preliminary experiments reveal inhibition of R. sphaeroides CcO by estradiol.The discrepancy between the results of the present work and that of Buhrow et al.  could bee and in  may probe and in   becausea et al. ,23. As s hormone . Indeed,−7–10−6 M for the total [−10–1.2 × 10−8 M for free testosterone and ca. 2x10−8–5x10−7 M for free estradiol [i values of 10−5–10−4 M determined for the hormone action on CcO in our experiments. However, it is noted that steroid hormones are very lipophilic and partition from water to octanol with a coefficient of 103–104 [4 brings the values of the order of 10−8 M (the free hormones in the blood serum) to the hormone concentrations in the mitochondrial membranes close to the estimated Ki (ca. 10−4 M). Therefore, quite noticeable inhibition of cytochrome oxidase by physiological concentrations of the steroid hormones looks possible.An obvious important question is whether the inhibition of the isolated CcO by steroid hormones may be relevant to physiology. The first point to be concerned is the hormone concentration range. Physiological concentration of the steroid hormones in the blood serum is around 10he total , ca. 1 ×stradiol ,26 that  103–104 , so that 103–104 . Thus, t50% values are cited for inhibition of cell growth by anabolic steroides: testosterone, 100 µM; androstenedione, 375 µM; nandrolone, 9 µM; norandrostenedione, 500 µM; norandrostenediol, 6 mM. Effective concentrations of tens to hundreds of micromoles are also given in the rest of the works mentioned above.At the same time, our data may be relevant to the experimental studies of steroid hormones action in vivo, studies with whole animals or (and particularly so) with cell cultures. Concentration of the hormones employed in such studies often reach a submillimolar or even millimolar range ,31,32,33The second point as to the physiological relevance of our data is that inhibition of the isolated CcO by steroid hormones has been observed under conditions rather far from physiological. For instance, all the data have been obtained in the presence of a detergent dodecyl-maltoside and it remains to be established whether the inhibition by the hormones can take place in case of the membrane-bound enzyme. Experiments with intact mitochondria and liposome-reconstituted CcO are required in order to resolve this question.c = 1.47 mM and 1.3 mM, see Finally, we would like to discuss the role of DM in the inhibition of CcO by steroids. Experiments with both testosterone and estradiol suggest that DM competes with the steroid hormones for binding with the enzyme. Notably, the close values of affinity between DM and CcO are determined independently in the two cases of inhibition . From a


Idiopathic generalised epilepsy (IGE) refers to a group of epilepsies resulting from the activation of neurons in the whole brain. This study aimed to evaluate the metabolite changes in thalamus as diagnostic biomarkers in IGE patients compared to healthy individuals using magnetic resonance spectroscopy (MRS) technique.t-test between the groups.The MRS was performed on 35 IGE patients (26 women and 11 men) with average age of 32 (ranged from 18 to 43) and 35 healthy individuals (13 women and 22 men) with average age of 31 (ranged from 21 to 50) as the control group. The levels of N-acetylaspartate (NAA), creatine (Cr) and choline (Cho) were measured using MRS. The NAA/Cr and NAA/Cho ratios were calculated for all participants. These values were statistically compared using The NAA had significant lower values in IGE patients, 9.6 (SD = 0.8) and 9.9 (SD = 0.7) for right and left thalamus, respectively, compared to 10.9 (SD = 0.9) and 10.7 (SD = 0.9) in control group. The Cr values in the left side of thalamus were significantly higher in IGE patients (6.7 [SD = 0.8] versus 5.8 [SD = 0.5]); however, there was no difference in right thalamus. Measurements showed no difference for amounts of Cho between the groups in both sides of thalamus. The NAA/Cr ratio was 1.48 (SD = 0.14) and 1.48 (SD = 0.16) for right and left thalamus, respectively, in IGE patients in comparison with 1.83 (SD = 0.2) and 1.86 (SD = 0.26) in controls. There was no meaningful variation between the NAA/Cho ratio of the right and left thalamus among the groups.Thalamic NAA, Cr and NAA/Cr ratio values in IGE patients showed statistical differences compared to healthy individuals. Evaluating metabolites variations in thalamus using MRS is suggested for differentiating IGE patients from healthy individuals. A seizure is a process that occurs as a result of abnormal, excessive and synchronous electrical depolarisation of a group of central neurons in brain. The diagnosis of epilepsy is made when seizure attacks are repeated due to a chronic background process .Idiopathic generalised epilepsy (IGE) refers to a group of epilepsies resulting from the activation of neurons in the whole-brain . This tyMagnetic resonance imaging (MRI) has become one of the useful diagnostic techniques for high-resolution structural imaging in epilepsy . MagnetiThe thalamus plays a central role in cortical synchronisation   and axial T1-weighted three dimensional spoiled gradient recalled acquisition in the steady state (GRASS) images  = 50°, FOV = 24 cm with 124–156 slice partitions of 1 mm thickness and two number of excitations (NEX). T2-tirm dark fluid images with TI = 2027 ms, TR = 6000 ms, TE = 88 ms and FA = 150° were obtained. The voxels of interest (VOI) were adjusted to individual anatomy on images to cover the right thalamus, right cerebellum, prefrontal cortex and occipital cortex based on a protocol proposed in a previous study by Savic et al. .MRS with the point-resolved spectroscopy-chemical shift imaging (PRESS-CSI) technique was performed as following parameters: VOI = 3.35 mm × 2.8 mm × 2.8 mm, NEX = 2, number of repetition (NR) = 15, scan time = 6 min + 28 s, TE = 99 ms, TR = 4900 ms, TI = 2027 ms and total examination time = 44 min. Raw data and images were stored on the MRI system computer. These data were transferred to another computer for subsequent analysis. These routine MRI images were used as a positioning matrix for measuring spectrogram and for determination of the VOI positions. In The magnetic field homogenisation was done manually at the beginning of every protocol. Water suppression pulses were applied automatically and the pre-saturation bands were placed around the VOI area in order to remove fat and extraneous signals from the tissues around the VOI. The obtained spectral data was processed by the computer software and the results were evaluated based on the area under the curve of metabolite peaks. The metabolite concentrations in absolute units of mmol/L (mM) for the NAA, Cr and Cho were measured based on the MRS spectrum analysis. The metabolite concentrations values were analysed using the free nuclear magnetic resonance-spectra calculation using operators (NMR-SCOPE) software . This software has the ability to analyse the peaks of all metabolites .Post-processing of the raw MRS data for obtaining the 32×32 profiles, followed by a mild Gaussian k-space filter and an inverse Fourier transformation for both water-suppressed and unsuppressed MRS data. Artifacts present in the time domain water-suppressed signal due to static magnetic field inhomogeneities and time-varying gradients were corrected by dividing the water-suppressed MRS signal by the non-water-suppressed signal , a procet-test in the IBM SPSS software version 11.5 . Statistical significance was assumed as P-values < 0.05.The NAA/Cr and NAA/Cho ratios resulted from the right and left thalamus in IGE patients and control group were calculated and compared using independent P-value = 0.01). The Cr values in the left side of thalamus showed remarkable higher values in IGE patients ; however, there was no difference in Cr values in right thalamus between IGE patients and healthy individuals. Amount of Cho showed no difference between the groups in both sides of thalamus.Thalamus metabolites including NAA, Cr and Cho were measured in both sides in IGE patients and control group. Mean and standard deviation (SD) values, the comparison of these metabolites, and NAA/Cr and NAA/Cho ratios are shown in P-value ≤ 0.001). The average NAA/Cr ratio in the right and left thalamus in healthy participants were higher in comparison with those sides of thalamus in IGE patients.The SD values of the NAA/Cr ratios in the right and left thalamus in both IGE and healthy participants were measured and presented in This study also evaluated the NAA/Cho ratios in the right and left thalamus. The results of the study showed that there was no significant difference between the NAA/Cho ratios in the IGE patients and control groups .We found that changes in thalamus metabolites  are the appropriate diagnostic biomarkers for differentiating IGE patients from healthy individuals using MRS technique.In a study by Savic et al.  using a In the current study, the multi-voxel methodology was used and the metabolite ratios in both side of thalamus were measured. In SVS technique, a single spectrum was acquired from a definite volume of tissue as an integral , 15, 17.P-value < 0.01) compared to healthy individuals. Since NAA is measured in a voxel, a reduction in NAA reflects a loss of nerve cells or axons, as well as neural damage and a disorder in body metabolism (The results of our study demonstrated that the value of NAA and NAA/Cr in the thalamus of IGE patients is significantly lower (tabolism .In this study, some evidence was found about the relationship between the metabolic changes and neural activity of thalamus in IGE patients as the main foundation of seizure generation in this type of epilepsy. Nevertheless, it was not determined whether the thalamus functioning disorder is specific to IGE or not. This is because in general, no structural damage was found in routine MRI images as a foundational area for epilepsy focus. The analysis of the spectrogram obtained through multi-voxel MRS method showed a remarkable decrease in the NAA and NAA/Cr ratio in the thalamus of IGE patients. We also found that the Cr value was higher in IGE patients in comparison with healthy individuals just in the left side of thalamus. This metabolite usually was used as internal reference, since it is the most stable cerebral metabolite. Therefore, higher values of Cr in IGE patients can indicate the neural problems. Previous studies , 12, 16 In a study, Fojtiková et al.  reportedAccording to the findings of this study and matching them with the clinical data of patients, thalamus plays a key role in the generation of seizures in IGE which means the presence of thalamo-cortical anomalies as a pathophysiologic foundation for IGE.In this study, different time and locations for gathering the MRS data were the limitations. Furthermore, differences in therapeutic modalities, like various pharmacotherapy programs, may affect the results of our study.Regarding the results of this study and previous researches , 16, 17,Our results demonstrated certain altered metabolite values in IGE patients compared to healthy individuals. Thalamic NAA, Cr and NAA/Cr ratio values showed significant differences in IGE patients. Therefore, MRS of thalamus in patients is suggested for differentiating the IGE patients from healthy individuals.


The numbers of unique sequences from organisms reported from soil, sediment, ice, aquatic, marine, animal, and plant (probably pollen) sources also peaked in this region, suggesting that this was the most biologically active region. The confluence of the high numbers of organisms, physiologies, and metabolic capabilities suggests the presence of energy and nutrient sources in the eastern half of the embayment. Data from the main basin suggested a cold oligotrophic environment containing fewer organisms. In addition to bacteria, both the basal ice and accretion ice contained sequences from a diverse assemblage of eukaryotes, as well as from bacteria that are known to be associated with multicellular eukaryotes.A combined metatranscriptomic and metagenomic study of Vostok (Antarctica) ice core sections from glacial, basal, and lake water accretion ice yielded sequences that indicated a wide variety of species and possible conditions at the base of the glacier and in subglacial Lake Vostok. Few organisms were in common among the basal ice and accretion ice samples, suggesting little transmission of viable organisms from the basal ice meltwater into the lake water. Additionally, samples of accretion ice, each of which originated from water in several locations of the shallow embayment, exhibit only small amounts of mixing of species. The western-most portion of the embayment had very low numbers of organisms, likely due to biologically challenging conditions. Increasing numbers of organisms were found progressing from west to east, up to approximately 7 km into the embayment. At that point, the numbers of unique sequences and sequence reads from thermophilic, thermotolerant, psychrophilic, and psychrotolerant organisms increased dramatically, as did sequences from alkaliphilic, alkalitolerant, acidophilic, and acidotolerant sequences. The number of unique and total sequences were positively associated with increases in concentrations of Na However, it is covered by 4 km of glacial ice, has an estimated temperature that hovers just below 0 °C, and has been continuously ice covered for approximately 14–15 million years [Subglacial Lake Vostok is an extreme environment that appears to support a diverse ecosystem concentrated in a shallow embayment on the southwestern corner of the lake ,2. Of thon years . It delion years ,9. BecauThe glacier contains several distinct types of ice that have been formed by different processes . At VostInitial hypotheses proposed that Lake Vostok was sterile due to the extreme and oligotrophic conditions. However, as information from geochemical, biological, and biochemical studies has accumulated, the results have suggested the existence of a complex biological ecosystem in the lake waters and sediments ,13,14,15Vostok 5G ice core sections from each of four regions were obtained from the NSF-ICL : glacial ice 2149 m), basal ice (3501 m and 3520 m), ice from the west region of the shallow embayment , ice from the middle of the embayment , and ice from the east region of the embayment . Four Vostok 5G ice core sections  were analyzed in a previous study 49 m, bas.2, 6 mM DTT (dithoithreitol), 330 μM dNTPs , and 100 units of SuperScript II reverse transcriptase  for each sample; incubated at 37 °C for 90 min and then placed on ice. Second strand synthesis consisted of 25 mM Tris-HCl (pH 6.9), 93 mM KCl, 4.6 mM MgCl2, 140 μM β-NAD+, 9.3 mM (NH4)2SO4, 0.5 mM dNTP mix (as above), 1.2 mM DTT, 5 U Escheria coli DNA ligase, 20 U E. coli DNA polymerase I, and 1 U E. coli RNase H ; incubated for 2.5 h at 15 °C. Next, 5 U T4 DNA polymerase was added to each sample and incubated at 15 °C for 7 min, and then placed on ice. The reaction was stopped by the addition of EDTA to a concentration of 30 mM. Then, each sample was subjected to chloroform/isoamyl alcohol (24:1) treatments, centrifuged at 14,000× g for 2 min to separate the layers, and the nucleic acids (aqueous phase) were precipitated in 0.5 M NaCl and 80% ethanol. Precipitation was performed at −20 °C for 15 min. After centrifugation at 14,000× g for 20 min, the pellets were washed with -20 °C 80% ethanol, and centrifuged at 14,000× g for 2 min. After decanting, the pellets were dried in a vacuum centrifuge drying  at 45 °C for 10 min, and then were rehydrated in 9 μL of DEPC-treated water. These were mixed with the corresponding DNAs. The isolated RNA was used to synthesize cDNA employing a SuperScript II kit  according to the manufacturer’s instructions. First, random hexamer primers were added to each 5 μL (RNA plus DNA) sample to a concentration of 6.5 ng/μL. Each sample was then mixed gently, incubated at 70 °C for 10 min, and then chilled on ice. The first strand synthesis consisted of 33.3 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 1 mM ATP, 100 pmols EcoRI (NotI) adapters (AATTCGCGGCCGCGTCGAC), 14 mM DTT, and 2.5 U T4 DNA ligase ; incubated at 15 °C for 16 h, and heated to 70 °C to stop the reaction. Next, 15 U T4 polynucleotide kinase were added, and incubated at 37 °C for 30 min. To stop the reaction, the samples were heated to 70 °C. Sephracyl size fractionation columns (supplied with the Superscript II kit) were rinsed with TNE (10 mM Tris (pH 7.5), 0.1 mM EDTA, 25 mM NaCl), and used to collect 23 fractions (35 µL each), each of which was subjected to PCR (polymerase chain reaction) amplification (20 mM Tris (pH 8.3), 10 mM KCl, 10 mM (NH4)2SO4, 0.8 mM dNTPs, 2 mM MgCl2, 0.4% Triton X-100, 10 mg bovine serum albumen, 1 U Taq DNA polymerase , 1 μM EcoRI (NotI) primer (AATTCGCGGCCGCGTCGA), and 5 µL of each DNA/cDNA sample, in a total volume to 25 µL. The thermocycling program was 94 °C for 2 min followed by 45 cycles of 94 °C for 1 min, 55 °C for 2 min, 0.1 °C/sec ramp to 72 °C, and 72 °C for 2 min. Once the cycles were finished, the samples were incubated at 72 °C for 10 min before being held at 4 °C. All amplified fractions were subjected to electrophoresis on 2% agarose gels  at 5 V/cm in TBE , 0.5 μg/mL ethidium bromide, and visualized with UV irradiation to determine concentrations and size distributions. Adapter sequences were added to the ends of each of the DNAs and cDNAs in order to amplify them in subsequent steps. They were added in the following reaction: 66 mM Tris-HCl (pH 7.6), 10 mM MgClEcoRI primer pair, and 1 µL of previously amplified DNA/cDNA samples in a total volume to 25 µL. The samples were purified using a QIAquick PCR purification kit , and quantified on 2% agarose gels (as above). Fractions 3–13 from each sample were used for further processing, based on having DNA distributions between approximately 100 and 1000 bp. Next, a total of five pairs of primers were synthesized. Each contained one of a set of multiplex identifier (MID) sequences on the 5’ end of the primers , and the EcoRI/NotI sequences (AATTCGCGGCCGCGTCGAC) on their 3’ ends. The primers were used in PCR reactions (as above) with 1 μM corresponding to each MID 2, 10 mM DTT, 1 mM ATP, 0.4 mM dATP, 0.4 mM dCTP, 0.4 mM dGTP, 0.4 mM dTTP), end repair enzyme mix . Samples were then incubated for 20 min at 25 °C, then 10 min at 70 °C, and held at 4 °C. Ligation of the Ion Torrent adapters was accomplished by adding NEB T4 ligase buffer for Ion Torrent, Ion Torrent Primers , NEBNext Bst 2.0 WarmStart DNA polymerase, and T4 DNA ligase . This was incubated for 15 min at 25 °C, then 5 min at 65 °C, and finally held at 4 °C. Each was precipitated with 0.5 M NaCl and 80% ethanol at −20 °C for 1 h. The nucleic acids were pelleted by centrifugation in a microfuge, and dried in a vacuum centrifuge  at 45 °C for 10 min. Each was rehydrated in 50 µL 1 mM Tris (pH 7.5), 0.1 mM EDTA.Ion Torrent adapters were ligated to the ends of the amplicons (from the previous step), using an NEBNext for Ion Torrent Library Preparation Kit . Each sample was prepared as follows: 1 ug DNA sample, end repair buffer (50 mM Tris-HCl (pH 7.5), 10 mM MgClA reaction for each sample was performed with 20 µL of the products from the step above, 1 uM of the corresponding pair of primers , and E is the EcoRI/NotI sequence), and 50 µL of the NEBNext Q5 hot Start HiFi PCR master mix . The thermocycler program was 98 °C for 30 sec followed by 15 cycles of 98 °C for 10 sec, 58 °C for 30 sec, 65 °C for 30 sec and once complete, the samples were incubated at 65 °C for 5 minutes, with a 4 °C hold. Once polymerization was complete the samples were purified with QIAquick columns using the previous procedure. Each was rehydrated in 1 mM Tris (pH 7.5), 0.1 mM EDTA, and quantified by 2% agarose gel electrophoresis (as above).Two samples were prepared for sequencing at the University of Pennsylvania Core Sequencing Lab . Sample one consisted of a mix of 150 ng of product from the negative control (sterile RO water), 150 ng from the 2149 m ice core section sample, and 150 ng from the 3501 + 3520 m ice core section samples, for a total of 450 ng in a volume of 50 µL. The second sample consisted of a mixture of 225 ng of product from the 3540 + 3569 m ice core section samples, and 225 ng from the 3585 m ice core section sample, for a total of 450 ng in a final volume of 50 µL. The sequences were received from the University of Pennsylvania Core Sequencing Lab as two files in FASTQ format and converted to FASTA format using Biopython (Open Bioinformatics Foundation). Only high-quality reads were used. The files were then divided in silico into their original five groups based upon the MID sequences within the primers , basal (3501 + 3520 m), west embayment (3540 + 3569 m), and east embayment (3585 m) data sets, sequence data from our previous research, including data from the middle of the embayment (3563 + 3585 m) and the western region of the southern main basin (3606 + 3621 m) samples , by searching NCBI information, publications, and other online resources. Species and/or strains were used exclusively to deduce ecological and physiological affinities. Determinations were made only when they were unambiguous. Organisms were then categorized into groups based upon gene identities (from BLAST), ecology, source, physiology, metabolic capabilities, and other notable species characteristics. Additionally, the data were used to compile information on metabolic pathways, such as nitrogen cycling and carbon fixation. Metatranscriptomic and metagenomic data from refs. ,2 were r+, K+, Ca2+, Mg2+, Cl−, SO42−, non-purgeable organic carbon (NPOC), and total amino acids  were modelled as fixed effects and the type of habitat was modeled as a random intercept. Alpha was set at 0.05, 2-tailed.Associations between the number of species  and crom ref. ) were stNational Center for Biotechnology Information (NCBI) sequence accession numbers are as follows: 2149 m - SAMN12175388; 3501 + 3520 m - SAMN12175389; 3540 + 3569 m - SAMN12175390; 3585 m - SAMN12175391. The BioProject that contains information related to the samples listed above located at the NCBI sequence read archive can be retrieved using the accession number PRJNA552298. The amount of sequence data for each sample was as follows: 2149 m  = 4571 bp; 3501 + 3520 m  = 46,421,725 bp; 3540 + 3569 m  = 50,689,858 bp; and 3585 m  = 71,241,841 bp. −6 from all samples, was 2644 , and they were limited to the basal ice (3501 + 3520 m) and the western and middle sections of the shallow embayment . Ice from this region was composed primarily of type 1 accretion ice. The number of unique and shared sequences for each sample indicated that each sample contained a majority of sequences that were unique to that sample  from the 2149 m sample is consistent with other studies that have reported low concentrations of cells at this depth of the glacier . TherefoH. salinarum R1 and H. salinarum NRC-1) mentioned above  contained a diverse set of organisms , including large numbers of Bacteria (407 unique sequences at ≥97% identity) and Eukarya (103 unique sequences), plus two of the Archaea  in the 3540 + 3569 m sample was much lower (130 at ≥97% identity) than those found in the basal ice sample (513) and the adjacent deeper middle embayment accretion ice sample, 3563 + 3585 m 1214). This portion of the accretion ice has large concentrations of suspended particles , Mg2+ , SO42− , and Cl− . Thus, from the western section of the embayment to the eastern side of the embayment, levels of Na+, Mg2+, SO42−, and Cl− increased, peaking around the 3570 to 3575 m depths  from m depths , followe−7 and 10−6, mol/L respectively), and higher in the accretion ice between 3670 and 3605 m , and decreased rapidly to approximately 10−8, and 9 × 10−7 moL/L (respectively) by 3620 m. There were positive associations between the numbers of species and concentrations of Na+ (P = 0.045), Ca2+ (P = 0.016), Mg2+ (P = 0.014), SO42− (P = 0.015), total amino acids (P = 0.004), and NPOC (P < 0.001). As these concentrations increased, so did the number of species that were observed.In addition to ion concentration comparisons, total amino acid concentrations and non-purgable organic carbon (NPOC) concentrations also were compared. Each of these was lower in the accretion ice at 3540 m , diatoms, haptophytes, and plant pollen in several of the ice core sections from the shallow embayment [Saprolignia parasitica, and a sequence closest to Notothenia coriiceps (rockcod), a ray-finned fish common in the Southern Ocean near the shores of Antarctica, that has antifreeze proteins and lives at water temperatures between −1 and 4 °C.For the eastern-most portion of the shallow embayment, represented by the 3585 m sample, the number of unique sequences was about 50% lower than in the sample from the middle of the embayment , which was within a region where Na+ concentrations were roughly half that of the peak values in the middle section of the embayment (approximately 3570 m), and where Ca2+ concentrations were low, had the highest number of halophilic/halotolerant species. These organisms may have been best suited to moderate Na+ concentrations.The relative numbers of psychrophiles and psychrotolerant species is high in the basal ice, which was expected, but these drop in the western section of the shallow embayment. The declines in most categories of organisms might be due to the extreme conditions that probably exist there. This includes the scraping of the glacier and embedded rocks on the lake-bed, local thawing and freezing, and a transition from basal ice meltwater to lake water, each of which exhibit differences in ion concentrations . There i regions . HoweverThe large numbers of sequences from thermophiles and thermotolerant peaked in the eastern half of the embayment . This poThe peninsula that separates the embayment from the main basin is approximately 10 km from the western shore . The iceThe BLAST results indicated that the vast majority of organisms in all samples were closest to heterotrophic species . The midAll of the steps in nitrogen cycling were represented in the sequences of organisms and genes found in the basal ice and in the accretion ice from the shallow embayment . This inMycobacterium spp. and Streptomyces spp.), nitrification (Mycobacterium spp. and Streptomyces spp.), denitrification , assimilation (most organisms), and decomposition . Proteobacteria are potentially responsible for the other portions of nitrogen cycling , although a few members of the Firmicutes  may contribute to this process. While the total number of Actinobacteria is higher in the accretion ice samples compared to the basal ice sample, the numbers and proportions of members of the Proteobacteria and Bacteroidetes active in the nitrogen cycle predominate in the accretion ice samples . However, there are some Actinobacteria potentially active in the nitrogen cycle in the accretion ice samples, including Frankia spp., Micromonospora spp., Nocardia sp., and Streptomyces spp.The communities of organisms indicated from the sequences in the basal and accretion ice suggests representation in vital metabolic pathways. However, different sets of organisms were indicated in the ice core sections for analogous processes. For example, members of the Actinobacteria predominate in the basal ice, and may be responsible for nitrogen fixation , were found only in the basal ice sample. This might indicate the presence of these components in the bedrock around the lake. Conversely, microbes that have metabolic capabilities for manganese compounds  were found only in the accretion ice. Manganese compounds often are found in areas of hydrothermal activity, which is another indication that hydrothermal activity might exists within the shallow embayment.Metabolic systems dealing with iron and sulfur were common among the species indicated by sequences from the basal and accretion ice samples. This was expected because of the nearly universal importance of compounds containing these elements within most organisms. These reactions were found within members of the Acidobacteria, Actinobacteria, and Proteobacteria. Bacteria capable of C-1 metabolic processes (members of the Alphaproteobacteria and Betaproteobacteria), as well as those that have pathways for chromium and uranium metabolism (e.g., Based on the sum of the limnological studies from the Vostok 5G accretion ice core section, Lake Vostok appears to contain a complex ecosystem, which is concentrated near the suspected hydrothermal activity in the eastern region of the shallow embayment. The conditions and organisms in the embayment differ greatly from those found in the region of basal ice. The basal ice sequence data indicates that there exists a community of at least 513 distinct species , of whicThe community of organisms within the embayment of Lake Vostok forms a more complex and diverse ecosystem that than the one indicated in the basal ice . The wesIn addition to a wide range of bacterial species, many eukaryotic sequences were present, as well as some bacterial species that are normally associated with eukaryotes . The basThe accretion ice had more unique sequences from eukaryotic species than did the basal ice, which suggested a more complex ecosystem in the lake . This in


Palaeocave sites in South Africa are world renowned repositories for palaeontological and archaeological material, dating from the terminal Pliocene to the Early Pleistocene. Due to their antiquity, complex karstification history and multifaceted infilling phases, palaeocave sites are notoriously difficult to contextualise. Further to this, 19th century lime-mining and diverse excavation and sampling techniques, have complicated stratigraphic interpretations of fossil-bearing deposits within the region. Locating and assessing newly discovered, minimally disturbed palaeocave sites allow for contextual information to be gathered with greater confidence and can aid in constructing a more robust understanding of the South African fossil record. Here, we use Drimolen Makondo; a minimally lime-mined ∼2.61 Ma palaeontological site, to apply a series of in-depth stratigraphic and micromorphological studies. Contextual data presented within this study, testifies to a relatively rapid infill with greater fluvial activity when compared to adjacent deposits at the younger ∼2.04–1.95 Ma Drimolen Main Quarry. The quantity of articulated macromammalian remains, high density of micromammalian remains and pollen identified, also highlights Drimolen Makondo as a key site for ongoing palaeoenvironmental studies at the Pliocene to Pleistocene transition in South Africa. The Fossil Hominid Sites of South Africa ‘UNESCO World Heritage Area’  Valley sites of Sterkfontein, Swartkrans and Kromdraai B have yielded the bulk of Early Pleistocene hominin remains from South Africa . All thrsensuex-situ and their stratigraphic location has been reconstructed, or assumed , and mechanical ; or biogenic  . High fr assumed . As suchTraditionally, the stratigraphy of the classic CoH sites  has been defined by a lithostratigraphic approach  with different lithologic units defined as ‘Members’ . ‘MemberWhile stratigraphy is a fundamental concept understood by archaeologists and palaeoanthropologists, understanding it dictates an in-depth understanding of the site’s formation processes, as the site is an active part of the landscape . This isThe aim of this paper is to provide the first comprehensive stratigraphic and micromorphological interpretation to reconstruct the site formation history of palaeocave deposits, as well as interred fossil material at Drimolen Makondo (DMK). The discovery of DMK demonstrates the potential for locating fossil sites that have not undergone extensive mining in the CoH. To date, only preliminary stratigraphic and micromorphological data is currently published for DMK. Due to the well exposed sections, minor mining damage and its spatial association with Drimolen Main Quarry DMQ: , assessiThe Drimolen fossil site  is located between ∼4–6 kilometers north of the Blaauwbank Valley sites  and posiExploration uphill of DMQ, identified a possible extension of the palaeocave system to the west within a small makondo . Subsequin situ across both decalcified and calcified deposits  Permit ID 2035. Macroscopic characteristics of sediments and stratigraphic relationships between facies were observed and described on natural profiles , as well as on recent excavation surfaces of decalcified material and exposed mining surfaces. Areas of excavation were constrained and named according to solution cavities or shapes of adjacent breccia walls . Fieldwo Remnant .All field samples were extracted from exposed profiles by hammer and chisel or angle grinder. Laboratory thin section preparation followed in situ breccia and its decalcification products. Excavations have also exposed dolomite bedrock in the far western area of the site and on ridges separating multiple dissolution channels. These channels were originated by ceiling collapse/dissolution over former cave passages that are aligned predominantly WNW  is a term used to define manganese hydroxide deposits ; p. 423,Multiple spatially distinct speleothem deposits are observed throughout DMK profiles. The first is the basal flowstone, which partially overlays residual wad along the base of the North West Remnant. The continuation of this basal flowstone can also be seen at the base of Main Makondo, at the base of the eastern profile . The conSpeleothem observed at the far north western portion of the site  overliesThe original talus cone at DMK is well-represented by the coarse blocks observed at the base of Main Makondo . This maTCB facies is typically composed of unsorted dolomite and chert clasts from pebble to large boulders (<30–40 cm) within a reddish-brown, silty loam matrix . Clast sMicroscopically, the fine component of TCB has a well-developed granular microstructure with loose granules . HoweverThe South East Breccia  is a spaBreccia units preserved above the TCB in Main and Eastern Makondo, and within the Eastern Remnant, Western and Southern Makondo, Chris’s Gym and the northern exposures of Maddie’s Bath are all characteristic of reworked talus and fluvial deposits and derive from erosion, transport and deposition of karst fill. Thus, collectively these facies are referred to as SBC, however several sub-facies that pertain to distinct phases or to local perturbations of fluvial activity across multiple sections see , have alsensuIn general terms, these SBC facies consist of interbedded coarse and fine sub-facies, particularly moving eastward , to mixed coarse (SBC), to fine sediments (SSS). Consequently, the SSS units are also well preserved around the upper exposures of the SBC facies . Fine leThe same features observed microscopically within SBC facies are also observed within distal SSS units towards the western exposures of DMK . HoweverA primary feature of the central talus cone at DMK, is the abundance of well-preserved partially articulated fossils within SBC, and to a lesser extent, TCB facies. The highest density of these remains has been recovered from a recess between the cave wall and TCB in the NE section of Eastern Makondo, where an entire articulated ribcage of a carnivore was discovered . SolutioPodocarpus were readily identified . Cyclical in-wash events promoted the formation of multiple fining upwards layers within SBC facies, observed within multiple profiles –16F. LayThe sedimentologic properties of the SSS facies are similar those of the SBC; the grain-size is however much finer, with fining-upwards layers starting with very fine gravel/coarse sand and typically terminated by dense, fine silt and clay accumulations. These sediments indicate a cyclic medium- to low-energy in-wash within the fluvial depositional environment followed by pooling. Thus, SSS is the product of low-energy erosion and surface transport of the distal talus sides  , providiMetridiochoerus specimen (DNM 57) located at DMK, which likely derived from a Stage 1 Metridiochoerus andrewsi has only been found in sites pre- 2.61 Ma  directly adjacent to the deposit, bioturbation and/or root penetration, and pollen observed microscopically within multiple profiles, can all be associated with a shallower chamber. Thus, a relatively short infilling time-span centred on 2.61 Ma is presented.Australopithecus afarensis, the emergence of robust- australopiths Paranthropus, and larger brained Homo lineage sometime after 2.7 Ma (DMK is a rare South African example of a Pliocene-Pleistocene boundary aged fossil site. This time period in which DMK is set (∼2.61 Ma) is palaeoenvironmentally significant, as it aligns with the onset of a long-term trend toward aridification in eastern Africa  and morer 2.7 Ma . Changesr 2.7 Ma .Australopithecus and the first occurrence of Paranthropus robustus, Homo erectus and archaeology at DMQ, and perhaps also Swartkrans Member 1 Lower Bank   , both of2.58 Ma) , and pot2.58 Ma) ; as well2.58 Ma) . This is2.58 Ma) , and woo2.58 Ma) . First, an coast . In thesan coast . The modan coast . From anentified . These mentified , furtherin situ. The richness in fossil remains including articulated fossils, coprolites and micro-mammals are also unique, and well-preserved in both the breccia facies and within the decalcified material. Lastly, DMK is now well confined as an australopithecine-aged site (∼2.61 Ma) that correlates with an ecologically significant time period associated with long-term aridification and variable climatic events, as well as first appearance and extinction of species. The high percentage of faunal remains as well as the microscopic observation of pollen grains, indicates DMK is a significant site for ongoing palaeoenvironmental studies.New advances in chronological methods have greatly aided contextualising notoriously complex karst deposits in southern Africa, however their application can only be effective with a comprehensive understanding of a site’s stratigraphy. In combining litho- and allostratigraphy, micromorphology and geochronology, this model deviates from the classic ‘Member System’ and has been effective in assessing the rapid depositional history at DMK. The importance in undertaking such a study at DMK is multifaceted. The first is that it provides an opportunity to assess an ancient palaeocave with intact stratigraphies, as the site has not undergone extensive speleothem mining. This has made stratigraphic interpretations more accessible, as multiple sequences within the site remain