A review of the literature on the anatomy of the lower female genital tract in therian mammals reveals, contrary to the general perception, a large amount of inter-specific variation. Variation in females is anatomically more radical than that in the male genitalia. It includes the absence of whole anatomical units, like the cervix in many Xenarthra, or the absence of the urogenital sinus (UGS), as well as the complete spatial separation of the external clitoral parts from the genital canal (either vagina or UGS). A preliminary phylogenetic analysis shows two patterns. Some morphs are unique to early branching clades, like the absence of the cervix, while others arose multiple times independently, like the flattening out or loss of the UGS, or the extreme elongation of the clitoris. Based on available information, the ancestral eutherian configuration of the external female genitalia included a cervix, a single vaginal segment, a tubular UGS, and an unperforated clitoris close to the entrance of the genital canal. The evidence for either bilobed or unitary glandes clitorides is ambivalent. Despite the wealth of information available, many gaps in knowledge remain and will require a community-wide effort to come to a more robust model of female genital evolutionary patterns. The phallus is a shared derived characteristic of amniotes and is thus of a single evolutionary origin . It is pIn the present contribution, we review the available literature on the anatomy of the female phallus, i.e., the clitoris, and place this knowledge into an anatomical and phylogenetic framework. Such comparative compilation is crucial for two reasons. First, the lack of comparative studies is the likely cause of inconsistencies in the terminology and anatomical interpretations further impeding scientific work and leading to poor understanding of female genital evolution and its causes. Second, comparative genital anatomy, when combined with other characteristics of mammalian reproduction such as ovarian cycle characteristics and social structure, forms the basis for understanding female genital function and sexuality, as well as their evolution. Our own interest in this area was sparked by the apparent correlation between the nature of the ovarian cycle and genital anatomy, in particular the type of ovulation and the presence of the urogenital sinus UGS: .Expanded comparative data on reproductive anatomy, when combined with species-specific reproductive physiology, will enable us to test hypotheses about female sexual function and evolution, based on the idea that evolutionary changes in sexual function will be reflected in changes in genital anatomy. A solid understanding of the anatomical evolution of female genitalia is thus a necessary condition for understanding the evolution of female sexual function and behavior.Sphenodon, aka Tuatara, a sister species of squamates . Classical anatomical literature , as wellquamates . This suorigin of direct sperm deposition into the female cloaca . The consequence of internal fertilization, however, is clearer: it emancipated amniote fertilization from the aquatic environment. The directionality of gamete deposition is overwhelmingly uniform—that is, in most species, the males deposit gametes into the female reproductive tract, where fertilization occurs (rather than the other way around as in seahorses). It, therefore, appears likely that the evolutionary origin and the early evolution of the phallus have been driven by its male reproductive function, although homologous structures are present in both sexes. The clitoral functions have thus likely arisen secondarily. Another interesting aspect is that in amniotes the intromittent organ arose as a Type I novelty , as well as its anatomical position are clearly homologous between male and female phalli. The two structures are indistinguishable during their early development, diverging relatively late in development , 2020At the beginning of phallus development, the urogenital system consists of a cloaca, connecting the endodermal extraembryonic allantoic sac and the gut . The mesvasa deferentia) connecting testes to the urethra. The resulting single duct, urethra, thus corresponds topologically to the UGS, and carries both urine and sperm. This pattern is conserved in male eutherians—the penis invariably envelops a single duct with joint function. Thus, despite the wide diversity of the late-developing penile characteristics , the integration between UGS  and penis appears unchanged across Eutheria, as reported in rich comparative compendium by The subsequent genital development involves the integration of the external genitalia with the inherently sexually dimorphic urogenital systems. In male therian mammals, this means the integration of the genital and urinary duct into the phallus. This duct is composed of direct derivatives of the mesodermal ducts , which give rise to genital tubes . The female genital tract connecting the ovaries with an external orifice instead develops from the paired mesodermal Müllerian ducts . This anatomical entity eventually differentiates into oviduct, uterus, cervix and potentially the proximal portion of the vagina. The developmental origin and evolutionary individuality of the vagina  are still disputed, even for humans. A commonly held model for human development e.g.,  is that The UGS, i.e., the confluence of urethra and the derivative of the Müllerian ducts, develops as the urethra, coming from the bladder, opens into the reproductive tract. However, the persistence of the UGS beyond embryonic stages, and its spatial relation to the clitoris are variable (see below).Given the large amount of interspecific variation in the anatomy of the female external genitalia in therian mammals, it is difficult to establish a terminology that can be applied to all species and anatomical configurations. In addition, the amount and detail of information available for different species is quite variable, with developmental information limited to a small handful of species. Thus, in many cases the meaning of the anatomical terms identified in the literature remains unclear. Furthermore, embryological units do not always correspond to adult morphological structures. As noted above, an example is the human vagina, to which two different embryological and genetic units are likely contributing, the distal (lower) and the proximal (upper) vagina. Keeping these limitations in mind, we propose a terminology that can be applied to most anatomical descriptions available in the literature.Corpus clitoridis. Most anteriorly, there is the transition from the uterus to the genitals, which is usually marked by the cervix, a quite distinct structure in most therian mammals. In Xenarthra  and in the afrotherian elephant shrews, the utero-vaginal canal, which topologically corresponds to the cervix and the vagina of other mammals, has a different structure , and the tructure , as will segment . Note th segment . Then thde of it .vaginal vestibule. Note that a tubular, longer UGS can also broaden at its caudal end, and is often described in the literature as vestibulum—we treat, therefore, information about the presence of vestibulum independently from the information on the presence of UGS. For instance, in humans the vagina is shorter than the vaginal segment as defined above  and the vestibule includes part of the vaginal segment and the UGS  the genital canal is longer than the penis. The genital canal is sometimes called the “vagina,” from a functional point of view, but such a term would hinder the evolutionary comparative work, as relative contributions to a genital canal of developmentally different parts differ across species.Further, we use a meta-term Labia minora. Prepuce is also referred to as clitoral hood as it is described in many species to cover the Corpus or Glans clitoridis.Phallus is furthermore associated with two skin folds, preputium (prepuce) and frenulum. Both are parts of Corpus cavernosum (CC) and the Corpus spongiosum (CS)  . The firsum (CS) , in addiIn contrast to the penis, the mammalian clitoris exhibits a wide range of structural and positional variation. Before delving into the systematic survey, we provide general phenotypic dimensions, along which female genitalia differ. These dimensions often do not represent overall evolutionary trends but rather are found repeatedly in various clades.Female external genitalia and their integration with the lower urogenital tract vary in multiple dimensions. One dimension is the relation between the genital and urinary tract. Ancestral adult mammalian anatomy, present in Monotremes and to some extent in Marsupials , involves the cloaca, the cavity into which genital and urinary tracts as well as rectum empty. In adult eutherians and at least some marsupials, the rectum is separated from the urogenital ducts. Variation persists in whether urinary and genital ducts also exit the body separately, or share some extent of the tract, forming the UGS with a single orifice . Where along the genital tract the confluence of the urinary and genital tract occurs is also highly variable, ranging from the very proximal part close to the utero-vaginal canal (which replaces the cervix) in Xenarthra, or very close to the end of the genital canal, or in a flat vaginal vestibulum . If the vaginal segment is considered as the proximal part of the genital canal extending from cervix to the urethral meatus, and the UGS as the distal part extending caudally of the urethral meatus, the relative length of UGS varies from a very long to a very short segment (vestibulum), or even complete absence. Together, the vaginal segment and (if present) the UGS, form the genital canal, if both are tube-shaped .Corpora cavernosa and the Corpus clitoridis are located ventrally along the urethra. The external Corpus clitoridis emerges either in the lumen of the UGS, the vestibulum, or on the rim of the vestibulum. As mentioned above, in many species , vagina and urethra open as separate orifices (no UGS), and parts of the Corpus and Glans clitoridis in this case are exposed on external body surface, ventrally to the urethral orifice. In a few mammalian species, spread among different clades, the Corpus clitoridis is traversed by either the urethra or the UGS and assumes a penis-like morphology. This often coincides with a hypertrophied clitoris, but clitoral elongation, up to the length of male phallus, also occurs in various further clades without embedding any of the ducts. As explained below, these elongated clitorides differ widely in structure, as well as in developmental origin.Further variability concerns the relation of the clitoris to the urethra and the UGS . ClitoraOs clitoridis  to some extent follows variation of the Os penis, the baculum , described in several species including human, form paired internal bulbs at the basis of the phallus, extend along the phallic corpus and build the glans at the tip of the phallus. This tissue is often associated with the urethra/UGS and in males provides a single column embedding the UGS  in penis and ensuring that the UGS remains open for ejaculation during erection. Other ligaments and muscles of the perineum are associated with the male as well as the female phalli and will be addressed below only where sufficient information has been found.Less well-documented is the variation in the presence and size of erectile bodies, in particular their internal parts. In general, the joined  ischium . They prGlans clitoridis. The morphology of elongated clitorides found in some species of Lophostoma bats has yet to be described.Overall, it is noteworthy that the clitoral parts have often been modified within single lineages and over short evolutionary times. This is suggested by the phylogenetic distribution and by the distinct anatomy involved in generating superficially similar phenotypes. A good example is the evolution of the particularly hypertrophied genitalia. These well-known large clitoral parts arise in some species of insectivores (moles), bats  will be addressed along with the clitoris, whenever available information permits.Didelphis virginiana; Sarcophilus satanicus; Corpus clitoridis is described as bifurcated, protruding in the UGS at its posterior end (opossum). Corpus cavernosum is described to fill the external part of the clitoris, and no information is available about the Crurae.The general anatomy of the marsupial clitoris appears to follow that of the penis: in the Virginia opossum , for which a true vagina has been reported  presents a bipartite (bilobed) clitoris with paired Crurae, bodies, and glandes  was described to have a simple small un-grooved clitoris on the vulvar edge  is hidden by large labia, often confused with a male penis. Also, the visible clitoris of Chlamyphorus (fam. Chlamyphoridae) is small. In contrast, the clitorides of Chlamyphoridae genera Euphractus, Tolypeutes, and Zaedyus are elongated, penis-like singular structures. However, the similarity to a penis does not extend to internal structures: in all cases, the urogenital meatus is located dorsally to the Corpus clitoridis at its basis, and the external Corpus clitoridisis often grooved on its dorsal side.The xenarthran  glandes . The paivar edge . Pocock var edge  also desT. tetradactyla), the clitoris is simple and undivided, covered by hairy labia (Cyclopes didactylus), the clitoris is similar to that of the Lesser Anteater, yet bilobed. Uterovaginal canal in Lesser Anteater is lined by simple columnar mucous epithelium, which turns into transitional epithelium of the UGS after confluence with urethra, thus lacking a vaginal segment  and Asian elephants . The Ischiocavernosus muscle , as well as in Squirrel monkey (Saimiri oerstedii), Capuchin monkey (Cebus capucinus), and Night monkey , in the latter with a well-developed prepuce. Similarly, Callimico goldii) a small external clitoris with bifid glans which is visible at the extreme ventral edge of the vulva. This species has a keratinized vagina of about 3 cm, covered with papillae, and ending in a vestibulum. On the ventral side of the vaginal/vestibular junction rises an elevation below which there is a urethral orifice  external genitalia of the (wild-caught) animals from various collections presents a more balanced impression of New World monkeys (NWM) genital anatomy . Wislock orifice . A recenpithecus ] also repithecus .Macaca mulatta) and situated in the ventral part of vestibulum. It is surrounded by a delicate prepuce and Labia majora, but lacks a frenulum (In Old World monkeys (OWM), the female external genital orifice is located close to anus, between the ischial callosities . This is in contrast to NWM, where the distance between the anus and the urogenital apparatus is sexually monomorphic. frenulum .Labia majora (the latter has been long disputed in apes). In Black-handed gibbon Hylobates agilis, the prominent sub-conical clitoris is clefted, reaching onto the ventral edge from the vulva and surrounded by the prepuce (Hylobates lar) and show a separate urethral orifice positioned in the vulva on the urethral prominence, dorsally to the clitoris and ventrally to the vaginal tube, thus lacking a tubular UGS. Authors also observed the menarcheal changes, mostly as enlargement of the labiae and external clitoral shaft and glans.Wislocki also briefly describes the external genitalia of apes, which have a distinct protruding external clitoris of moderate size and vulva surrounded by two fatty crescent cushions, likely  prepuce . Dahl an prepuce  describePan troglodytes), the clitoris is visible, reaching out from a very short vulva, and has a flattened form. The clitoris and genitalia of Orangutan (Pongo sp.) are described as similar to those of Chimpanzee. The mountain gorilla (Gorilla beringei) manifests a conspicuous, protruding clitoris with a well-developed prepuce, but without frenulum , which possibly refers to the M. bulbospongiosus. With the current data, the presence and evolution of bulbs remains an intriguing, yet open question.Human female genitalia are described in various general and specialized texts in detail e.g., . ParticuCrurae, which attach to the pubic bone with M. ischiocavernosus in both sexes, and this muscle is described as not smaller in human females than in males  has recently been reviewed extensively in comparison with the Os penis , a species known for presence of ovotestes and enlarged genital parts .The phylogenetic distribution of baculum; . The Os M. ischiocavernosus and M. bulbospongiosus.The mammalian pelvic floor includes many muscles, ligaments, and fasciae, involved in a large number and diversity of pelvic functions, such as locomotion, sphincter, and reproductive functions, but also the anchoring and support of inner organs. Many ligaments of the urogenital system are dimorphic between the sexes due to the dimorphism of the urogenital system they support, but also due to the divergent functionality, e.g., pregnancy. Rather than addressing the whole complex, however, in this review we report the information about those muscles of the pelvic floor which directly relate to genital function. A comparative review of the muscles of pelvic floor would, however, be strongly needed for functional understanding. Here, most attention will thus be paid to two muscles, Musculus ischiocavernosus connects the Crurae of the phallus with the ischium, and likely serves to stabilize erection in males. A second muscle, M. bulbospongiosus, runs circular around the basis of the penile/clitoral shaft  and is thought to maintain blood pressure, similarly thought to support male erection. The evolution of both muscles has been addressed by several classical anatomists. Musculus ischiocavernosus and M. bulbospongiosus are thought to both be derivatives of the M. sphincter cloacae. The precursor of the M. ischiocavernosus likely formed as a subset of fibers of the sphincter that attached to the wall of the cloaca and to the ventral rim of ischium, before the separation between rectum and UGS occurred. Concordantly, M. ischiocavernosus in all eutherians and some marsupials. For example, in female Quoll , cloaca is associated with M. ischiocavernosus, and Corpora cavernosa (in Crurae penis) are described already in marsupials. In Eutherian females with UGS such as felids, M. ischiocavernosus attaches to the tissue just ventral to the urogenital sphincter, where also Corpora cavernosa appear. In contrast, M. bulbospongiosus likely arose by muscular fibers separating from the external urogenital sphincter muscle after the urogenital and anal tracts had diverged  specific nomenclature often hinders recognition of evolutionary continuities.Like for other internal parts, detailed descriptions of the perineal muscles are rare, with some exceptions. Such is the description of domestic dog by http://vertlife.org), however, the branches are scaled for illustrative purposes and do not reflect temporal information. The character states and their coding are provided in With the information summarized above, we attempted a phylogenetic reconstruction to reveal the evolutionary changes in several characters –9. We usThe UGS, i.e., the genital canal posterior to the confluence of urethra and vagina, is highly variable in its length, if present at all. The phylogenetic reconstruction shows that a lengthy tubular type likely is the ancestral eutherian condition , with inWe defined the vaginal segment as the portion of the reproductive tract between the cervix and the urethral meatus . As xenaCorpus clitoridis is highly variable among clades , and is absent in all other eutherian clades and is thus likely ancestral in therians and lost in boreoeutherians (A bipartite structure of the therians .T. matacus), a true vagina and possibly cervix has been reported . Despite scarce information, the presence of internal clitoral roots is reported consistently enough to assume that these parts are generally present in therian females, as they are in males. This appears different for the Corpora spongiosa, the clitoral bulbs. These inner parts associated with the clitoris are rarely described, yet few authors explicitly mention their absence, which would indicate that an attempt was made to search for them. Bulbus spongiosum are generally present in female mammals  but show great variation in size. It is at this point uncertain whether this part is rare, very inconspicuous, or whether a lack of focus on this region has prevented an assessment of its phylogenetic distribution. In contrast, bulbs are a very well-developed part of the human female clitoral complex . Also, the variation appears greater in the early branching eutherian clades, in particular in Xenarthrans, than in Boreoeutheria and the later branching clades within Boreoeutheria, such as primates or rodents. In general, female external genital traits apparently possess high evolvability, manifested not only by high phenotypic variation, but also by independent origination of similar structures, using different evolutionary paths, such as is the case with hypertrophied clitoris or its associated parts. The same can also be said about the internal clitoral structures. Internally placed paired clitoral roots consisting of  be seen . Despiteless extreme morphologies. Third, and most important, comparative work requires identification of homologous parts, which can be difficult from descriptions of the female genitalia and perineal muscles. Whereas the female external genitalia and lower genital tract are of common origin in therian females, the definitions of their parts are sometimes derived from their spatial interrelationship , from their histology , or their function . Prioritizing one of the aspects is a statement about their relative rates of evolutionary change among the aspects. For example, if the location of urethral meatus relative to reproductive tract is considered most evolutionarily stable, we can consider the part from the cervix to the meatus to be the vagina , and the part from the meatus to the urogenital orifice to be the UGS, across all mammals. Would we encounter a species in which epithelium does not change consistently with this border, we may conclude that changes arose by epithelial migration, or the change of the epithelial morphology as adaptation to its function . Alternatively, prioritizing the histology as the most stable aspect, we may conclude that the urethra can open into the midsection of the vagina, and that we must reconsider the naming of the caudal part as UGS and, thus the criterion of the transition between the two. Similarly, if we define vagina by the function as a copulatory organ, this will include UGS in some species and not in others. In addition to these challenges, comparative approaches must overcome the presently inconsistent anatomical nomenclature across the extended history of anatomical studies.This compilation inevitably inherits the weaknesses of the original literature. Apart from the mentioned sparsity of anatomical information and small sample sizes per species, the weaknesses include undetermined phase of the ovarian cycle at the time of collection or observation, unbalanced taxon sampling, and sometimes unclear homology relations. First, the genitalia of many mammalian females change during the reproductive period of the cycle, hence adding to variation, although the general location of anatomical structures will not be affected. Second, there is understandably a bias in favor of research on species with unusual anatomy, such as the hypertrophied clitoris of the Spotted hyena females. The bias toward these extremes should not be taken as a sign of the common occurrence of such anatomies, but rather as the lack of coverage of Most of these challenges will be overcome by extending the range of research on female reproductive and genital anatomy, as appears to be well underway. In order to understand how the genital anatomy evolves in the context of its reproductive functions, it will be important to combine anatomical knowledge with neuroanatomy, physiology, and endocrinology, beyond the excellent works on genital function in humans and mice , 2008c.At the very least this overview should cast doubt on the widely held view that “diverse male genitalic morphology” contrasts with “uniform female morphologies”  and showicac026_Supplemental_FileClick here for additional data file.


The role of gypsum level on the long-term strength and expansion of soil stabilised with different lime contents is not well understood. This research, therefore, studied the effect of varying gypsum concentrations of 0, 3, 6, and 9 wt%  on the performance of sulfate soil stabilised with two lime levels (4 and 6 wt%). This was carried out to establish the threshold level of gypsum/lime (G/L) at which the increase in G/L ratio does not affect the performance of lime-stabilised sulfate soil. Both unconfined compressive strength (UCS) and expansion, along with the derivative thermogravimetric (DTG) analysis, were adopted to accomplish the present objective. Accordingly, the result indicated that the strength and expansion were proportional to the lime and sulfate content, of which a G/L ratio of 1.5 was the optimum case scenario for UCS, and at the same time, the worst-case scenario for expansion. This discovery is vital, as it is anticipated to serve as a benchmark for future research related to the design of effective binders for suppressing the sulfate-induced expansion in lime-stabilised gypseous soil. Soil stabilisation by use of cementing agents is one of the conventional techniques used to obtain engineering materials having superior properties ,2. This Gypseous soils are widespread and covered about 20% of the land in the world, with a gypsum content varying from very low (h (≥50%) ,14,15. Th (≥50%) . The sweh (≥50%) ,18. Therh (≥50%) .To avoid the damages associated with gypsum dissolution and soil swelling, engineers traditionally tend to use a combination of chemical and mechanical soil stabilisation techniques ,16. This≥ 10.5) . The ett≥ 10.5) ,25,26.In the past, several research studies have identified that the onset of problems associated with ettringite formation depends on the sulfate content, lime content, and moisture content in the soil . For exaTherefore, in this current study, an attempt has been made to investigate the effect of two lime levels (4 and 6 wt%) on the stabilisation of artificial sulfate kaolin soil made with varying gypsum levels . Both physical (volume change) and mechanical (change in UCS) properties have been assessed to complement the present objective in order to establish the threshold of G/L for superior swelling and strength performance. The mechanism of alteration of lime-stabilised gypseous soil was also brought out by performing a thermal analysis (TG/DTG) on both expansion and strength specimens.The kaolin used in this study was semi-processed kaolin in the form of a fine powder with a pH of 5.37, a specific gravity of 2.14 The mineralogical analysis revealed that its dominant minerals are kaolinite mineral and quartz, whereas the oxide composition analysis shown in lsewhere  suggesteyl group ,33.The sulfate type used in this study was gypsum in the physical form of a fine white powder with a percentage purity of ng soils ,35,36,37The lime used in this study was quicklime (CaO) in the physical form of an off-white powder with a pH of 12.6, a relative density of 3.31, a specific gravity of 2.82  calcite ,33.A total of eight mix compositions see  were desThe chosen lime contents were in agreement with the typical binder range used by other researchers ,18. ThesFor clarity, the sample mix code shown in At the preliminary phase of laboratory experimentations, it was essential to establish the optimum moisture content (OMC) and maximum dry density (MDD) of stabilised soil. Therefore, proctor compaction tests were conducted in accordance with  to deterformance ,46,47, aFor specimen preparation, enough dry materials see  capable lsewhere . SubsequThe unconfined compressive strength test was performed on three specimens per mix proportion, in accordance with ,49. The ing with . ThereafThe degree of swelling was established by means of linear expansion measurement, in accordance with . ImmediaThe perspex cells were then covered with prefabricated lids, equipped with dial gauges to measure the vertical displacement  of the specimens. On the completion of the initial reading recordation, the water was carefully added to the perspex cells, through the upper inlet, using a siphon. This was performed to ensure a minimum disturbance of the compacted specimens. Followingly, the level of water was carefully increased until the exposed bottom part of the specimens was submerged in water, and such a water level was kept constant to ensure that no water evaporation from the specimens occurred. Finally, the process of soaking was monitored daily for a period of 200 days. Ultimately, the mean of the change in vertical length of the two specimens was reported as the expansion of a particular mix composition.The thermogravimetric and derivative thermogravimetric (TG and DTG) analysis was performed on randomly powdered pieces collected from the fractured UCS and swelling specimens using a TA instruments TGA55 kit, which was manufactured by Waters Ltd and obtained from TA Instruments - a subsidiary of Waters Ltd, Cheshire, UK. A test specimen weighing about 5 mg was placed in an alumina crucible, and then the test analysis was operated from room temperature up to 1000 The unconfined compressive strength of kaolin specimens made with various gypsum amounts  and stabilised with two lime levels (4 and 6 wt%) is presented in By comparing both 4L- and 6L-based series, it was evident that a mix composition of K–6G–4L (G/L = 1.5) demonstrated the highest 90-day UCS of 2105 The typical expansion trends, over a longer soaking period of 200 days, for kaolin specimens made with different gypsum concentrations  and stabilised with two lime dosages (4 and 6 wt%), are shown in Like the case of UCS, it was observed that the control formulations (without gypsum) tended to show the lowest expansion during the whole soaking period, reaching an ultimate expansion of 4% for K–0G–4L and 5% for K–0G–6L. This expansion occurred immediately after soaking in water and reached the ultimate magnitude within the first week. However, this expansion trend was not the case in the presence of gypsum, as there was a staggering expansion phenomenon, and this phenomenon was proportional to both the lime and gypsum content. In this context, the expansion (4%) of K–0G–4L was increased gradually to 14, 24.2, and 24.9%, while the expansion (5%) of K–0G–6L was increased to 14.8, 24.6, and 30%, as the gypsum increased to 3, 6, and 9 wt%, respectively. By comparing both systems (4L and 6L), it can be also inferred that there was a fixation gypsum content at which a further increase in gypsum content had no significant contribution to the ultimate expansion magnitude. This fixation gypsum dosage depended on lime content, of which 6 and 9 wt% of gypsum were the worst-case scenarios for 4 and 6 wt% lime, respectively.The DTG curves of kaolin soil specimens dosed with 0, 3, 6, and 9 wt% of gypsum and stabilised with 4 and 6 wt% of lime at two different curing conditions (7 days of moist curing and 200 days of soaking) are presented in As seen in After 7 days of moist curing, the ettringite peak was enlarged as the gypsum content increased, and such an enlargement was more pronounced at higher gypsum and lime contents. This implies that the ettringite amount is directly proportional to gypsum and lime contents. The increasing ettringite peak was also in line with the observation of the UCS trends, suggesting valuable causativeness for the variation in strength. Furthermore, the gypsum peak was relatively invisible in the case of K–3G–4L and K–3G–6L, suggesting that 3 wt% of the gypsum can be consumed in the formation of ettringite during the 7-day moist curing period. As for the portlandite peak, it has only appeared in the case of the control mix of K–0G–6L, the existence of which indicates a sign of the uncomplete consumption of lime during the 7-day moist curing period.Upon a prolonged (200 days) water soaking period, the DTG curves revealed considerable changes in the thermal stability of the stabilised specimens. In this regard, no trace of portlandite peak was noticed, demonstrating the importance of moisture and curing age on the completion of lime hydration. Moreover, the ettringite peak was slightly increased, relative to the unsoaked counterparts, for all the formulations, while gypsum moved contrariwise and even disappeared in some cases. At 4 wt% lime, the gypsum peak was completely disappeared in K–3G–4L and relatively invisible in the case of K–6G–4L. On the other hand, at 6 wt% lime, the gypsum peak was only detected in K–9G–6L, with a very negligible peak height. The disappearance of gypsum can be ascribed to the abundance of water molecules within the system, which tend to completely solubilise the unconsumed ingredients and act as an avenue for ion migration, hence becoming a source of ions supply at the nucleation sites of ettringite. As for the appearance of gypsum trace, this could be attributed to the excess of gypsum amount over that of the soluble sulfate threshold (gypsum/lime ratio of 1.5) consumed during the formation of ettringite.The above laboratory investigations brought out the influence of the variation of lime and gypsum contents on the expansion and strength performance of kaolin clay. It was observed that the strength and expansion of kaolin soil were improved on the application of lime, with more improvement being recorded for a higher lime content (6 wt%). This improvement is believed to be because of two basic reactions: short team reactions (cation exchange and flocculation/agglomeration of soil particles) and long-term reactions (pozzolanic reactions) ,52. WhenIn the absence of sulfate, the divalent calcium cation fixes to the surface of soil particles, replacing the available exchangeable cations ,18. The  cations . This re cations . The red cations ,55, whic cations . This chBesides the flocculation/agglomeration of soil particles, the hydroxyl ions (resented . The pre10–to–13 . This inl[OH]4−) . The ratl[OH]4−) .Following the dissolution of clay minerals, the pozzolanic reactions thereafter take place between the excess lime and dissolved (Al and Si) ions from treated soil . These pIn the presence of gypsum, however, an alteration in the short-term reactions (cation exchange and flocculation/agglomeration of soil particles) and long-term reactions (pozzolanic reactions) can occur due to the dissolution of gypsum . This isUnder the sealed condition (moist curing condition), the growth of ettringite is controlled due to the lower dissolution ratio of gypsum and the unavailability of enough water for dissolving gypsum . TherefoHowever, under the soaking condition, the movement of water within the stabilised soil tends to solubilise the unconsumed ingredients and acts as an avenue for ion migration, facilitating the diffusion of reactants . TherefoOverall, the result of the laboratory experimentations reported in this paper suggests that the strength and expansion performance of lime-stabilised kaolin were proportional to gypsum (G) and lime (L) content, of which a G/L ratio of 1.5 was the optimal case scenario for the UCS and the worst-case scenario for the expansion. This is of great importance and practically pertinent to all the civil, geotechnical, and geological engineers/researchers involved in the development of an effective binder. This discovery is vital, as it is anticipated to serve as a benchmark for future research related to the design of effective binders for suppressing the sulfate-induced expansion in lime-stabilised sulfate soil.The addition of lime to kaolin soil in the absence of gypsum increases the compressive strength and reduces the expansion through the change of fabric  and the formation of pozzolanic reactions. However, in the presence of sulfate, lime can yield a higher strength magnitude, but it also induces a drastic expansion due to the formation of an excessive ettringite.The consumption of gypsum during the nucleation and growth of ettringite in lime-stabilised gypsum-bearing soil is a function of lime content, moisture content, and curing condition.The presence of sulfate (gypsum) increases the compressive strength and the expansion of lime-stabilised soil as the gypsum content increases, with a more pronounced effect at a higher gypsum content and higher lime content. This is because the amount of formed ettringite is a function of both lime and gypsum contents. Under the sealed condition, the ettringite improves the strength through the reduction of porosity and interlocking of the matrix, while under the water soaking condition, it causes expansion due to its higher water absorption capability.The strength and expansion performance of lime-stabilised soil were directly proportional to the gypsum (G) and lime (L) content, of which a G/L ratio of 1.5 yielded the optimum condition for the UCS and the worst-case scenario for the expansion.The main conclusions emerging from the results of the laboratory experimentations reported in this study can be drawn as follows:


This report describes the molecular characterization of a serotype O foot‐and‐mouth disease virus (FMDV) recovered from a field outbreak in the Zambezi region, Namibia during July 2021. Sequence analysis demonstrates that this FMDV belongs to the O/EA‐2 topotype sharing closest nucleotide identity (99.5%) to FMD viruses collected since 2018 in Zambia. This is the first detection of serotype O in Namibia, and together with the cases that have been recently detected in southern Zambia, represent the first time that this serotype has been detected in the Southern African FMD endemic pool since 2000, when a virus of Asian origin (O/ME‐SA/PanAsia) caused an outbreak in South Africa. This incursion poses a new threat for the region and the potential onward spread of O/EA‐2 will now need to be closely monitored since serotype O vaccines are not widely used in Namibia, nor in neighbouring countries. The disease circulates in Asia, Africa and parts of South America where outbreaks lead to livestock production losses and disruption to international trade  is caused by a highly contagious virus (FMDV: family https://www.wrlfmd.org/southern‐africa/namibia#panel‐4082).Namibia maintains a ‘FMD‐free without vaccination’ zone officially recognized by the World Organisation for Animal Health (OIE), located to the south of the Veterinary Cordon Fence (VCF). The VCF initially established in 1897 to protect farms in the south of the country from rinderpest, now provides a barrier to protect the high‐value commercial export markets in southern Namibia from FMD that circulates in the eastern Zambezi region  in the northern part of the country. The Northern Communal Areas of Namibia have no official OIE status for FMD. FMD outbreaks detected recently in the north of Namibia include continued cases due to serotype SAT 2 (topotype III) in 2021 as well as outbreaks due to serotype SAT 3 (topotype II) in 2019  and Zambia (2018–21).n = 7) collected on 15 July 2021 from Nfooma were submitted to the OIE Sub‐Saharan Africa Regional Reference Laboratory (SSARRL) for FMD  where FMDV diagnosis was carried out as previously described  was then added and incubated at 37°C and monitored for cytopathic effect (CPE) for 48 h. Samples were considered negative for virus isolation if no CPE was observed after the third passage. An in‐house sandwich ELISA using serotype‐specific rabbit and guinea pig antisera for FMDV serotypes O, A, SAT 1, SAT 2 and SAT 3  involved suspect FMD cases in cattle herd of 144 animals  where 100 (69.4%) of the animals showed clinical signs of FMD including lameness, salivation and frothing of the mouth, blisters in the mouth and hooves. Further suspicion of FMD was subsequently raised in cattle (on 13 July 2021) at Nfooma located in Katima Mulilo Urban  where a total of 70 out of 87 (80.5%) animals exhibited clinical signs indicative of FMD. Vesicular epithelium samples (The 1D nucleotide sequences encoding VP1 (633 nucleotides) were compared with other related serotype O viruses collected from the region. In addition to published sequences, the comparative data included unpublished sequences recently obtained at the FAO World Reference Laboratory for FMD  for 33 samples received from Zambia. Collection locations and dates for these samples are outlined in Table The complete VP1 encoding nucleotide sequences were aligned using BioEdit 7.0.5.3 Hall,  and Clus3Testing at BVI, Botswana, yielded FMDV positive results for five out of seven of the Namibian samples using virus isolation which were all characterized as serotype O using antigen ELISA. The sequences encoding VP1 of four selected virus isolates from Namibia  were identical to each other and were categorized as belonging to the EA‐2 topotype within serotype O. This analysis showed that the Namibian sequences shared closest nucleotide identity (99.5%) to two FMDV isolates that had been collected from the Western Province of Zambia during 2021 (O/ZAM/9/2021 and O/ZAM/11/2021; Figure The O/EA‐2 topotype originates from East Africa (FMD endemic Pool 4; Paton et al., Since 2018, O/EA‐2 viruses within a new genetic clade have spread across Zambia to cause outbreaks in Central, Copperbelt, Lusaka, North‐Western, Southern and Western Provinces Figure . The prePhylogenetic analyses Figure  highlighThe detection of O/EA‐2 in the Zambezi Region located in the Caprivi Strip in north‐east Namibia represents the first time that serotype O has been detected in the country. This incursion now poses a new threat to the FMD endemic region of Namibia and other countries in the region, such as Angola, Botswana and Zimbabwe. Botswana also maintains an OIE official FMD‐free without vaccination zone in the south, but like Namibia has no OIE status for the northern part of the country. The introduction of a new serotype or an antigenically novel variant can pose challenges for regional FMD control initiatives as was recently documented by the rapid spread of the A/ASIA/G‐VII lineage in Asia (Bachanek‐Bankowska et al., In summary, these findings highlight a new FMD threat for countries in Southern Africa and motivate field teams and laboratory scientists to increase surveillance within the region to rapidly detect further outbreaks due to this topotype. Now that this serotype has been identified, new resources will be required to ensure that laboratories in the region can correctly identify new clinical cases due to serotype O as well as detect serotype O‐specific antibodies in sera collected as part of surveillance activities. Furthermore, FMD vaccines selected for use in the region should now consider these risks, and vaccination campaigns may need to absorb the extra costs of adding a serotype O antigen component to the vaccines that are deployed.The authors declare no conflict of interest.


The COVID–19 pandemic has caused considerable interest worldwide in antiviral surfaces, and there has been a dramatic increase in the research and development of innovative material systems to reduce virus transmission in the past few years. The International Organization for Standardization (ISO) norms 18,184 and 21,702 are two standard methods to characterize the antiviral properties of porous and non-porous surfaces. However, during the last years of the pandemic, a need for faster and inexpensive characterization of antiviral material was identified. Therefore, a complementary method based on an Inactivated Virus System (InViS) was developed to facilitate the early-stage development of antiviral technologies and quality surveillance of the production of antiviral materials safely and efficiently. The InViS is loaded with a self-quenched fluorescent dye that produces a measurable increase in fluorescence when the viral envelope disintegrates. In the present work, the sensitivity of InViS to viral disintegration by known antiviral agents is demonstrated and its potential to characterize novel materials and surfaces is explored. Finally, the InViS is used to determine the fate of viral particles within facemasks layers, rendering it an interesting tool to support the development of antiviral surface systems for technical and medical applications. The trigger is the beta-coronavirus SARS-CoV-2, which is responsible for various symptoms such as inflammation of the throat and respiratory tract, coughing, shortness of breath, fatigue, fever, myalgia, conjunctivitis, loss of smell and impaired taste. In severe cases, it can lead to acute lung failure, multi-organ failure and death2. Although effective vaccines and better treatment strategies have been developed in the meantime, new highly contagious mutations and low vaccination rates in poor countries continue to force the health care systems to their limits. Therefore, non-pharmaceutical interventions such as contact reduction, hygiene and facemasks remain important to keep the propagation of the virus at bay3.As of June 2022, the number of confirmed COVID-19 cases worldwide has reached 529 million and the number of deaths has risen to over 6 million4. However, also other mechanisms of disease transmission are also possible. For example, aerosol transmission can occur indoors in crowded and poorly ventilated spaces, and it is also possible to catch COVID-19 indirectly through touching surfaces or objects contaminated with the virus from infected people, followed by touching the eyes, nose or mouth (fomite transmission)4. It has been shown that SARS-CoV-2 is stable from a few hours to days depending on the chemistry of the surface on which the virus is deposited. For example, van Doremalen et al. demonstrated that SARS-CoV-2 remained stable on plastic and stainless steel surfaces for several hours. Viable virus could be detected up to 72 h after application to these surfaces, and the half-life of SARS-CoV-2 was estimated to be 5.6 h on stainless steel and to 6.8 h on plastic5. In contrast, no viable virus could be detected on cardboard surfaces after 24 h, and copper surfaces tended to inactivate the virus within 4 hours5. Chin et al. confirmed that SARS-CoV-2 is more stable on smooth and hydrophobic surfaces. While no infectious virus could be recovered from printing and tissue culture papers after 3 h, viable and infectious virus was still present on treated smooth surfaces (glass and banknotes) after 2 days, on stainless steel and plastic after 4 days and on the hydrophobic outer layer of a surgical mask after 7 days6.The overall goal of these measures is to slow down disease transmission between people. COVID-19 spreads mainly by respiratory droplets among people who are in close contact with each otherSince this high surface stability increases the risk of smear infection, it would be highly beneficial if virus particles that reach vulnerable surfaces such as touch screens, doorknobs, ventilation filters, textiles, train seats or handrails could be inactivated immediately without further disinfection processes. The same applies also to virus particles deposited on facemasks, since inappropriate mask handling is a commonly faced problem.Therefore, the development of antiviral materials, coatings and facemasks is of tremendous importance not only to fight COVID-19, but also to prevent similar pandemic or epidemic outbreaks in the future.10, radiation such as ultraviolet light with wavelength between 200 and 300 nm (UVC)13, thermal treatment like autoclaving15, oxidation17 or high positive surface charge19. When new antiviral materials are developed, it is crucial to investigate and quantify their potential to trap, inactivate and/or kill the viruses. Currently, there are two ISO norms available, which regulate the measurement of antiviral activity on plastics and other non-porous surfaces (ISO 21702) and the determination of antiviral activity of textile products (ISO 18184). In both norms, the infectious virus titer needs to be determined by either a plaque assay or the Median Tissue Culture Infectious Dose (TCID50) method. Both of these assays require working with the real infectious virus and are based on the principle that cytopathic effects caused by the virus can be visibly assessed in vitro. Although these assays deliver reliable and comparable results on the antiviral activity of the materials, they have several disadvantages: working with real viruses such as e.g. SARS-CoV-2 requires special infrastructure (e.g. biosafety level 3 equipped labs) and trained employees. This prevents the widespread use of these analyses in most industrial and research settings and consequently delays the rapid development of novel antiviral materials as observed during the current COVID-pandemic. Consequently, novel characterisation methods are urgently needed to facilitate the fast, cheap and safe pre-screening of a large number of materials and surfaces for potential antiviral properties.There are several ways to inactivate or even destroy viruses. The most known are detergents or ethanol solutions which disintegrate the lipid and protein structure of the virusIn this work, an inactivated octadecylrhodamine R18 loaded A/Brisbane 59/2007 influenza virus system (InViS) is exploited as a surrogate for an infectious virus to assess viral disintegration by simple fluorescence measurements. This novel virus system allows the study of antiviral effects of different chemicals, cleaning agents and materials in a simple way: the fluorescence increases when virus particles disintegrate, since the fluorescent probe is self-quenched inside the intact virus lipid structure , different potential antiviral nanoparticles (NPs) and surface coatings. In addition, we exploited the use of InViS to assess and quantify the fate and localization of virus particles in facemask layers.20 are established procedures applied in vaccine research. Here, we explore the potential of such an inactivated virus in the field of antiviral characterization. The Inactivated Virus System (InViS) used in this work consists of an inactivated octadecylrhodamine (R18) labeled A/Brisbane/2007 influenza virus solution, whose fluorescence increases upon membrane rupture or fusion21.The inactivation of viruses and loading with fluorescent dye13 (± 9.8 × 1010) particles mL−1 and the virus particles exhibited a very homogeneous size of 110.6 ± 0.8 nm. The fluorescent label inside the virus was self-quenched and the fluorescence of the intact virus was only 23% of that after addition of the detergent octaethylene glycol monododecyl ether (OEG)  only induced a partial release of the R18 label, while an almost complete release similar to the positive detergent control was detected upon addition of 70% ethanol.To verify that InViS is not only sensitive to detergents, but can also detect other known antiviral compounds with different antiviral potency, we performed further experiments with 70% ethanol and citric acid (1 M). Both chemicals induced a significant increase in fluorescence signal intensity Fig. . However2), gold (Au), silver (Ag), selenium (Se) and graphene oxide (GO) possess antiviral properties29, yet the underlying toxicity mechanisms are often not fully understood. To investigate if the antiviral mechanisms of these NP types may include viral disintegration, we incubated the InViS with different NP concentrations for 2 and 24 h and measured the fluorescence intensities to detect the potential release of the fluorescent R18 label. None of the investigated NPs were able to destroy the InViS envelope, since fluorescence levels did not increase and were comparable to the negative control (intact InViS without NPs) , zinc oxide (ZnO), titanium dioxide (TiO30). The InViS was brought in contact with either the coated surface or the liquid form of the coating solution and a significant increase in fluorescence intensity could be detected in both cases  need to exhibit strong antiviral activity. We used a filtration efficiency test system to expose textile community masks, surgical masks and FFP2  masks to aerosols containing InViS. As a comparison, filtration efficiency testing was also performed with salt solutions, which represents the current European standard procedure (EN13274-7) to assess the filtration efficiency of textiles. After the filtration testing, the different mask layers were peeled apart and the localization of the particles was assessed by Scanning Electron Microscope (SEM)  and fluorescence measurements .Micrographs of the outer woven layer Fig. A,A',B anDuring the filtration efficiency experiments, the air was first passed through the pores in the outer woven layer of the masks, where a preferential accumulation of salt particles near the pores was observed while most of the outer layer fibers were devoid of particles Fig. B,B'. InV31.The severe COVID-19 pandemic has dramatically changed people's lives in the past 2.5 years. Things that used to be taken for granted, such as a regular job in the office, meeting friends, attending cultural events and travelling, were suddenly no longer possible. Lock-downs forced people all over the world to stay at home, and had a negative impact on the economy. Healthcare systems reached their capacity limits, and many people suffered or even died from a severe illness. To fight the pandemic, many researchers and clinicians worked under a high pressure to understand the disease and develop efficient vaccine and treatment options. In parallel, the textile industry intensively worked on the development of user-friendly, comfortable and effective facemasks to slow down disease transmission between peopleSoon, it was recognized that the development of antiviral materials and surfaces was recognized as an efficient mean to slow down the spread of SARS-CoV-2 and prevent infections by direct contact. However, the development of antiviral materials is a time- and cost-intensive process. One of the major issues was that the antiviral properties of newly developed coatings and materials could only be assessed by conducting tests with the real virus (ISO 21702 and ISO 18184). This required trained employees and a special infrastructure (e.g. biosafety level 3 equipped labs) which were difficult to access for most material developers and considerably delayed the development of innovative antiviral materials.32. The most interesting feature of InViS is that the fluorescent probe is self-quenched within the viral membrane. As a consequence, the fluorescence of InViS significantly increases when the virus particles disintegrate and release the fluorescent dye.Therefore, alternative test methods that are inexpensive as well as easy and safe to handle are highly valuable to facilitate the rapid pre-screening of novel antiviral material designs to advance innovation and rapidly identify promising candidates for further testing with the real virus. In this study, we present InViS, a novel alternative virus system that allows a fast, cheap and safe detection of virus disintegration activity of liquids, compounds and materials by simple fluorescence measurements. We used an inactivated Rhodamine-18 labeled A/Brisbane 59/2007 Influenza virus that has close structural similarities to SARS-CoV-2, namely constituting an enveloped RNA virus of ~ 100 nm in size with hemagglutinin on its surface. The virus has been inactivated with β-propiolactone, which preserves the antigenic virus structure, but renders the virus non-infectious due to alkylation of nucleic acid bases, suppression of genome replication, induction of genome degradation and protein and genome cross-linking35. Similarly, a low pH can lead to membrane degradation38. Therefore, we exposed InViS to 70% ethanol and citric acid to test its sensitivity against known antiviral liquids. Indeed, both chemicals led to a significant increase in fluorescence. When the InViS particles were in contact with the ethanol solution, complete viral disintegration occurred within 5 min. In the case of citric acid solution, the viral disintegration remained partial, indicating that the InViS system can deliver (semi-)quantitative data on virus disintegration.It is known that ethanol can disintegrate virus particles by lipid membrane dissolution and protein denaturation39. Therefore, we investigated whether Ag, Au, CuO, ZnO, TiO2 and graphene oxide NPs are able to disintegrate InViS. Although antiviral effects have been described for the investigated NP types, none of the tested NPs induced viral envelope disintegration even upon prolonged exposure for up to 24 h. We even observed a slight decrease in fluorescence signals with increasing NP concentrations, which could be due to particle adsorption to and/or penetration of the viral capsule surface and subsequent removal from the liquid sample during the centrifugation step. This would be in line with results published by Kim et al., where a co-precipitation of Au NPs and Influenza A virus during centrifugation was reported24.Because some NPs can have antiviral properties they are extensively explored for the development of novel antimicrobial materials and coatings23. NPs containing Cu could catalyze the generation of radicals via Fenton or Fenton-like reactions, oxidizing the capsid proteins and consequently blocking the viral infection at an early stage23. AuNPs have been shown to oxidize the disulfide bonds of the hemagglutinin glycoprotein on the viral surface, causing its inactivation and thus impeding the membrane fusion of the virus with host cells23. TiO2 NPs may damage lipids and glycoproteins in the viral envelope40. Graphene nanosheets were able to interrupt hydrophobic protein–protein interactions and graphene oxide could adsorb virus particles, therefore preventing their interaction with the cell membrane.These results for the NPs are consistent with the existing literature, which suggests that antiviral activities of most NPs appear to rely on other effects than capsule disintegration. For example, Ag NPs were shown to bind or denature the viral capsid protein or inhibit the virus from binding to cell receptors, therefore preventing virus entry into the host cells30) with InViS. We report a strong increase in fluorescence, and confirm that the coating solution resulted indeed in viral disintegration. Such rapid tests can be highly beneficial in the development of antiviral liquids and coatings. For example, they allow the screening of a large number of different substances, compositions and concentrations to identify the most promising solution for further development.With this knowledge, the biggest limitation of our InViS system becomes evident: membrane dissolution is not the only inactivation method and therefore not all antiviral materials can be characterized using InViS. Nevertheless, many antiviral materials rely on viral envelope disintegration. The aim of the InViS is not to replace the existing and approved ISO tests. Rather, it was designed to offer a simple, cheap and safe alternative to assess viral disintegration  at an early stage of research and development. The system is particularly interesting for researchers and manufacturers who want to assess the efficacy of materials that are designed to disintegrate the lipid envelope. During this study, we evaluated such an antiviral coating solution  for a fast, inexpensive and safe assessment of virus disintegration by simple fluorescence measurements. InViS can be further used to study the fate and localization of viral particles on non-porous as well as porous materials such as technical and medical textiles, rendering it a valuable tool to support the development of novel antiviral materials, coatings and facemasks.−1) determined by Single Radial Immunodiffusion Assay, SRID. Rhodamine B octadecyl ester perchlorate (R18) was purchased from Merck KGaA (The Netherlands) and dissolved in HLPC grade anhydrous ethanol at a final concentration of 10 mM. R18 solution (40 µL) was added to an influenza solution (1 mL) dropwise at room temperature (RT) under continuous stirring at 200 rpm for 15 min. Non-incorporated R18 was removed by separation on a Sephadex G50 column (Merck KGaA). The void volume fraction was collected and further characterized.Chemically inactivated and purified monovalent influenza virus A/Brisbane/59/2007 (H1N1) solution at GMP grade was obtained from Seqirus  hemagglutinin (HA) . To confirm that the virus remained intact after shipment, DLS measurements were performed to determine the hydrodynamic diameter of the virus particles and their polydispersity index in PBS before and after the addition of the OEG . Furthermore, fluorescence measurements of the serially diluted virus with and without OEG  were performed with a Horiba FluoroMax SpectraFluorometer to establish the detection limit.Particle size, size-distribution and concentration were analyzed by Nanoparticle Tracking Analysis on a Malvern NanoSight LM10 instrument. The sample was diluted 1:10,000 in HNE buffer (HEPES (10 mM) pH 7.4, NaCl (142.5 mM), EDTA (5 mM), filtered through a 0.1 µm syringe filter before use) and injected into the analysis chamber of the 405 nm laser module with a constant flow of 70 units at a controlled temperature of 25 °C and a viscosity setting of 0.975–0976 Cp. 5 captures were performed, with each capture having a duration of 60 s. Camera setting was level 15 with a detection threshold of 3. R18 fluorescence and fusion activity were determined to confirm fluorescence quenching and the presence of HA on the virus surface as described70% ethanol (CAS 64-17-5) and citric acid  were incubated with InVis (0.4% v/v). The samples were homogenized with a vortex mixer and incubated for 5 min at RT. Fluorescence measurements were performed using a Horiba FluoroMax SpectraFluorometer. The excitation wavelength was 560 nm and emission spectra were measured between 580 and 650 nm. An InViS solution in PBS (0.4% v/v) was used as a negative control to provide the fluorescence intensity of the intact virus. As positive control, OEG  was added to disintegrate the virus and indicate the corresponding fluorescence intensity.To study potential antiviral effects of NPs, we used particles that were used and fully characterized in previous studies. The most relevant properties of the particles are summarized in Table −1 and homogenized using a vortex mixer (1 min). Particles available as powder  were suspended in ultrapure water to a stock suspension of 1 mg mL−1 using a probe sonicator operating at 230 V/50 Hz  for 5 min at 13 W.Particles available as suspensions  were diluted with ultrapure water to a stock suspension of 1 mg mL−1) were incubated with a solution of InViS in PBS  for 2 and 24 h. The incubation was carried out at RT in the dark and with continuous shaking. The suspensions were centrifuged for 10 min at 4500×g to remove the NPs. Fluorescence of the supernatants was measured with a Horiba FluoroMax SpectraFluorometer. To quantify the antiviral effect, fluorescence was compared to the fluorescence of a virus suspension in PBS (negative control) and a virus suspension treated with OEG . To exclude autofluorescence or fluorescence quenching of the NPs, fluorescence of pure NP suspensions (without virus) and fluorescence of NPs incubated with virus and detergent were also measured.Different particle concentrations  and fluorescence measurements. To characterize the properties of the coated form, a volume of 0.5 mL was spread evenly in disposable petri dishes and incubated at RT for 24 h. The ISO norm 21,702, which covers the antiviral characterization of non-porous materials, was used as a guideline for sample preparation. The virus inoculum, 0.4 mL for each sample, was composed of a 20% v/v InViS stock solution in PBS. An inoculum with InViS (20% v/v) in PBS and an inoculum with InViS in PBS (20% v/v) and OEG (62.5 mg mL−1) in empty petri dishes were used as a negative and positive control, respectively. An inert polymer (low density polyethylene (LDPE)) film (40 mm × 40 mm) was used to cover the inoculum. The film was gently pressed down to form a sandwich structure and maximize the contact surface area between the inoculum and the antiviral sample, while preventing leakage beyond the edges of the inert film. The samples were stored for 24 h in the dark at RT. 20 mL of PBS were added to the petri dish and the dishes underwent agitation to ensure the homogenization of the freshly added PBS and the remaining inoculum. After mixing, 2 mL of the washout were collected and pipetted into transparent cuvettes. The emission fluorescence spectra of the washout was characterized using the Horiba FluoroMax SpectraFluorometer. The excitation wavelength was set to 560 nm and the emission intensity was measured between 580 and 650 nm. For each sample, two replicates were analyzed.An antiviral solution  was fed to the aerosol generator . As the virus stock solution was composed of virus in PBS, the aerosol solution contained 1% v/v of PBS. Therefore, not only InViS, but also PBS residues were present in the aerosol.To assess the fate and localization of InViS and salt particles in facemasks, the filtration efficiency set-up presented in Fig. After filtration efficiency experiments, the mask layers were peeled apart and analyzed separately to localize the InViS particles. First, scanning electron microscopy (SEM) images of the inner and outer layers provided a qualitative analysis of the particle presence on the mask fibers. For this, a Hitachi S-4800  SEM was used. Prior to imaging, the mask layers were mounted onto SEM stubs with a conductive double sided carbon tape and sputter coated with 7 nm of gold/palladium (LEICA EM ACE600) to reduce electron charging effects. The settings for SEM imaging were an accelerating voltage of 2 kV and current flow of 10 µA. Secondly, the amount of virus on each layer was evaluated by fluorescence measurements. For this, the different mask layers were placed in 8 mL of 70% ethanol for a period of 2 h. Afterwards, 2 mL of the solution were pipetted to transparent cuvettes and fluorescence measurements were conducted with a Horiba FluoroMax SpectraFluorometer .$p < 0.01, £p < 0.001 and #p < 0.0001.R was used for figures and statistical calculations. The statistical differences were assessed using the Student's t-test and the following symbols represent the corresponding p values: *p < 0.05,


The worldwide spread of chikungunya over the past two decades calls for greater knowledge and awareness of the virus, its route of transmission, methods of diagnosis, and the use of available treatment and prevention measures.Aedes mosquito-borne febrile disease, has spread from Africa and Asia to Europe and the Americas and from the tropics and subtropics to temperate regions. International travel is a pivotal influence in the emergence of chikungunya as a global public health threat, as evidenced by a growing number of published reports on travel-related chikungunya infections. The striking features of chikungunya are arthralgia and arthritis, and the disease is often mistaken for dengue. Although mortality is low, morbidity can be profound and persistent. Current treatment for chikungunya is supportive; chikungunya vaccines and therapeutics are in development. Travelers planning to visit areas where the mosquito vectors are present should be advised on preventive measures.Chikungunya virus infection, an Chikungunya is an emerging disease in the Americas. Frequent travel, the presence of at least two competent mosquito species, and a largely naïve human population in the Western Hemisphere create a setting conducive to future outbreaks. Awareness of the disease and its manifestations is critical to effectively and safely manage and limit its impact. Vaccines in late-stage clinical trials offer a new pathway to prevention. The COVID-19 pandemic has dominated the world of infectious diseases, and the world itself, since March 2020. As of October 2022, COVID-19 has claimed more than 6.5 million lives globally [An emerging disease on this list is chikungunya, a mosquito-borne viral infection that causes debilitating joint and muscle pain. The acute symptoms typically resolve within a week to 10 days, but in some cases arthritis and arthralgia persist for weeks, months, or even years in a condition mimicking rheumatoid arthritis –6, 7•. TWith the exception of mother-to-child transmission, chikungunya is not spread person-to-person •, 11•. MThis article will review the history and epidemiology of chikungunya, the clinical features of acute and chronic disease, the use of diagnostic tests, the available treatment options, and preventive measures. The pre-travel consultation for persons planning international travel is important in providing advice to reduce risk for the disease and to discuss the symptoms in returning travelers that raise concern for chikungunya.First recognized in what is now Tanzania in 1952 , 16, chiAedes albopictus, in many parts of Europe and the USA [Aedes albopictus in Italy had been documented in the early 1990s, in a warehouse of a tire retreading company, where used tires imported from the southeastern USA were infested with mosquito eggs. A second outbreak of locally transmitted chikungunya occurred in Italy in 2017 with approximately 400 total cases [Chikungunya virus caused sporadic outbreaks in Africa and Asia for decades but did not vault to public attention until an outbreak in coastal Kenya in 2004 spread to Indian Ocean islands and then to India, where it caused explosive epidemics ••, start the USA •. The pral cases •. While al cases .In late 2013, the first documented cases of locally transmitted chikungunya in the Western Hemisphere occurred in the Caribbean island of St. Martin. The disease then spread rapidly throughout the Caribbean and Central and South America •, 27. AsChikungunya among US travelers is a recent phenomenon ••, 51•. Chikungunya became a nationally notifiable disease in the USA in 2015. In that year, 896 cases were reported, all but one related to travel, with the highest number reported from California, New York, Florida, and Texas, where one case of locally transmitted disease occurred •. In subThe European Centre for Disease Prevention and Control (ECDC) collects data on chikungunya infections reported by countries across the European Union and European Economic Area . In 2014Clinicians should consider chikungunya in travelers who present with fever, rash, and arthralgia or arthritis and are returning from areas of the world with known chikungunya transmission, including the Caribbean , 56. In Chikungunya is an arthropod-borne virus (arbovirus) along with Zika, dengue, and West Nile. The virus exists as a single serotype  with thrAedes aegypti and Aedes albopictus mosquitoes, the same mosquitoes that transmit dengue and Zika virus. They typically bite, aggressively, during the daytime. Aedes aegypti has been described as a “nervous feeder”; if a blood meal is interrupted, it will move on to another person, producing clusters of infection within a household [The chikungunya virus is transmitted primarily by the female ousehold •. EpidemBlood-borne transmission has been documented and in utero or intrapartum transmission is possible but rare. The highest risk for maternal–fetal transmission occurs when an infected mother is highly viremic around the time of delivery ••. OtherHumans serve as the primary host of the virus during epidemics. Anyone with suspected chikungunya infection should avoid mosquito exposure for at least 7 days after the onset of illness to reduce the possibility of transmitting the virus to mosquitoes, which could then transmit to other humans. The complete transmission cycle from human to mosquito and back to another human can take place in less than a week . Once a Symptomatic disease develops in the majority of infected individuals, usually 2 to 6 days after the mosquito bite, with a range of 1 to 12 days ••, 62. AThe differential diagnosis of chikungunya includes many viral infections, particularly dengue, Zika, and other alphaviruses depending on geographic exposure  ••. GivenCardiac manifestations are of particular importance due to the high associated mortality –65, 66••The most common neurologic complication of chikungunya is encephalitis, occurring in the Réunion Island outbreak at a rate of 8.6 per 100,000 persons •. ClassiMost symptomatic chikungunya infections resolve within a week to 10 days, followed by a post-acute stage that may last for 3 months after the onset of illness. Beyond that time, up to 40% of patients may experience painful, incapacitating symptoms that persist for months or even years in a post-chikungunya chronic polyarthralgia or arthritis, often accompanied by chronic fatigue and depression , 75, 76•A systematic review of 34 studies found that 43% of cases did not fully resolve within 3 months after onset . FollowiPreliminary diagnosis relies on the individual’s clinical presentation and a thorough travel history. Chikungunya can be misdiagnosed as dengue and even Zika in areas where the viruses are circulating, as their clinical features overlap. Co-infections can occur •, 84–89 https://www.cdc.gov/ncezid/dvbd/index.html). Serological cross-reactivity can occur with other alphavirus infections, including Mayaro, o’nyong’nyong, Semliki Forest virus, eastern equine encephalitis, western equine encephalitis, and Venezuelan equine encephalitis. Investigators have also detected chikungunya virus in saliva, urine, and other bodily fluids in both the acute and convalescent phases [Confirming the diagnosis requires laboratory testing. The more widely available tests include polymerase chain reaction (PCR) to detect viral RNA in the first 8 days of illness, or acute-phase serology to detect IgM, IgG, and neutralizing antibodies toward the end of the first week of illness (> 4 days post-onset) paired with a convalescent-phase serology; although possible, viral cultures in the first 3 days of illness are less frequently used •, 92. Cot phases , 94.Immediate treatment of chikungunya infection is supportive and symptomatic, emphasizing rest, fluids, and relief of pain. Patients with suspected chikungunya should be managed as though they had dengue  until dengue has been ruled out, due to the increased risk of bleeding and potentially life-threatening hemorrhage associated with the use of aspirin and other NSAIDS. Once a diagnosis of chikungunya without dengue co-infection is confirmed, NSAIDS, corticosteroids, and/or physiotherapy may help relieve persistent joint pain. Treatment of chronic chikungunya arthritis will likely require rheumatology, physiotherapy, and pain management expertise •.A Latin American Consensus Conference held in 2017 issued a number of recommendations for the diagnosis and treatment of chikungunya-related inflammatory arthropathies •. In theA single-dose live attenuated vaccine candidate that has completed a phase III immunogenicity and safety trial in the USA •, with rA virus-like particle vaccine candidate; a phase II study employing one- and two-dose schedules showed a “robust and durable” serum neutralizing antibody response . Phase IA live recombinant measles-vectored vaccine candidate, which demonstrated “excellent safety and tolerability and good immunogenicity” in a phase II trial •, 102.Therapeutics and vaccines for chikungunya are in active development. Vaccine candidates include the following:Drugs being evaluated for use in treating chikungunya infection include antivirals currently licensed in the USA or elsewhere for other purposes, such as ribavirin, arbidol, suramin, favipiravir, and sofosbuvir –106. OthChikungunya virus infection has emerged as a global health threat over the past two decades, expanding to all continents except Antarctica. The disease has low mortality but high morbidity, including the risk of lifelong disability and its economic and psychosocial consequences. Widespread international travel and the presence of two competent mosquito vectors in most parts of the world create a setting for future outbreaks. Travelers heading to destinations where mosquitoes carrying the virus are endemic should seek pre-travel consultation. The emergence of chikungunya as a global public health concern calls for greater knowledge and awareness of the virus, its route of transmission, methods of diagnosis, and the use of available treatment and prevention measures •• see T.Table 3S


Many viruses utilize the lymphohematogenous route for dissemination; however, they may not freely use this highway unchecked. The reticuloendothelial system (RES) is an innate defense system that surveys circulating blood, recognizing and capturing viral particles. Examination of the literature shows that the bulk of viral clearance is mediated by the liver; however, the precise mechanism(s) mediating viral vascular clearance vary between viruses and, in many cases, remains poorly defined. Herein, we summarize what is known regarding the recognition and capture of virions from the circulation prior to the generation of a specific antibody response. We also discuss the consequences of viral capture on viral pathogenesis and the fate of the captor cell. Finally, this understudied topic has implications beyond viral pathogenesis, including effects on arbovirus ecology and the application of virus-vectored gene therapies. Limiting the amount of virus freely circulating in the bloodstream can be important for controlling viral pathogenesis and transmission. However, despite early advances, this field of study has become overlooked and understudied. Innate immune cells in the liver and spleen constantly survey and remove from circulation viral particles without the aid of virus-specific antibody. The details of these host–virus interactions, and the consequences thereof, remain unknown for many viruses. Yet, understanding this phenomenon has implications not only on bettering our understanding of disease progress, but also on arbovirus ecology and the development of effective virus-vectored gene therapies. It has been established for over a century that foreign particles introduced intravenously (i.v.) into vertebrates are rapidly removed from circulation . In 1904Since initial studies in the late 19th century, there have been great leaps in our understanding of microbial vascular clearance. However, most mechanistic studies focus on clearance of blood-borne bacteria, and limited mechanistic reports exist on viral vascular clearance. Herein, we introduce key cell types of the liver and spleen demonstrated to mediate the bulk capture of circulating virions prior to generation of a specific antibody response and summarize known host and viral mechanisms orchestrating clearance of specific viruses from mammalian circulation. We also discuss consequences of vascular clearance on viral pathogenesis and additional implications of these studies on both arbovirus ecology and virus-vectored gene therapies.Table 1). As an aside, it should be noted that these studies on viral capture from the bloodstream assume vascular dissemination occurs via free viral particles. However, hematogenous spread of some viruses, such as cytomegalovirus, primarily occurs in a cell-associated manner—which adds another layer of complexity -insensitive mechanism of clearance exists [Clearance of measles virus (MV)-like particles from murine circulation is rapid, with a half-life of 1 minute and undetectable plasma virus levels by 30 minutes pi . These ce exists .A series of studies by Mims in 1959 analyzed the serum clearance of mousepox virus ,49. By 2Building on this earlier work, a 2017 study on dissemination of vaccinia virus (VACV) found i.v. inoculation of low viral doses  unable to effectively disseminate to murine ovaries . HoweverNatural antibodies have been proposed to promote splenic uptake of VACV, as μMT mice  exhibited both decreased serum clearance of the virus and decreased titer of virus in the spleen . Howeverop/op mice . For example, s.c. inoculation of Semliki Forest virus  into thorotrast-treated mice produced accelerated and heightened viremia compared with untreated controls . Similartain KCs ,190) alstain KCs .Another method to investigate the impact of vascular clearance on disease severity is the utilization of specific viral mutants with altered clearance kinetics. One such mutation in the capsid of the hepatitis A virus (HAV) promoted faster serum clearance than WT virus due to its stronger affinity for glycophorin A expressed on erythrocytes . CompetiFollowing uptake of circulating viral particles, KCs restrict viral gene expression and replication in a manner dependent on signaling through the type-I interferon receptor (IFNAR). Upon VACV vascular clearance, viral replication was controlled by KC IFNAR signaling and promoted host survival . DespiteHowever, KC–virus interactions may also result in KC death. For example, while most AdV particles captured by the liver are degraded ,196, as Capture of circulating virions by the liver can affect development of an antiviral T-cell response . LSECs + macrophages elicited an inflammatory response [While splenic macrophages do not appear to play a dominant role in the physical removal of circulating virions , their participation does impact the immunological responses to infection. For example, murine MZMs and MMMs are potent producers of IFN-I in response to i.v. inoculation of UV-inactivated herpes simplex virus 1 ,115, whiresponse . Specifiresponse .+/CD11c−/Ly6C+/Ly6G+ cells [Meanwhile, several studies with AdV ,57 and VG+ cells . MMM-ampG+ cells ,56, 57, Table 1). The mechanisms orchestrating clearance vary between viruses, and in some cases, the RES may utilize multiple, redundant avenues to capture a virion, as seen with DENV [The literature contains a vast variety of papers on the kinetics of viral vascular clearance and subsequent biodistribution. If susceptible to vascular clearance, most virions are rapidly cleared from circulation by the liver, with some splenic participation  affects clearance of circulating viruses, although some studies observed impaired bacterial clearance in patients suffering liver cirrhosis , 207. El


Increasing nonzoonotic human monkeypox virus (MPXV) infections urge reevaluation of inactivation strategies. We demonstrate efficient inactivation of MPXV by 2 World Health Organization‒recommended alcohol-based hand rub solutions. When compared with other (re)emerging enveloped viruses, MPXV displayed the greatest stability. Our results support rigorous adherence to use of alcohol-based disinfectants. The global spread of human monkeypox virus (MPXV) has activated the highest alert level of the World Health Organization (WHO), which declared the virus a public health emergency of international concern  for surgical and hygiene hand disinfection in healthcare settings and to reduce transmission of pathogens  fetal calf serum, 1% (vol/vol) nonessential amino acids, 100 IU/mL penicillin, 100 μg/mL streptomycin, and 2 mmol/L l-glutamine. For preparation of MPXV, we seeded cells at a concentration of 0.33 × 10We confirmed the presence of MPXV by a 2-step, quantitative, real-time reverse transcription PCR (qRT-PCR). The first step determines the presence of orthopoxvirus-specific DNA by using panorthopoxvirus‒specific qRT-PCR, and the second step detects and differentiates MPXV clade I (former Congo Basin) and II (former West African) by using an MPXV-specific qRT-PCR. Isolate MPXV-DUS_001 was classified as MPXV clade II.We assessed virucidal activity of WHO formulation I and II, as well as ethanol and 2-propanol, based on European guideline EN14476 as described . We test>6.7 at concentrations of 60% and 80% (vol/vol) (>6.7 at concentrations of 60% and 80% (vol/vol)  , panel Avol/vol) , panel B>6.7 at concentrations >60% and >40% (vol/vol), respectively. A minimal concentration of 40% ethanol (vol/vol) nearly inactivated the virus completely (RF 6.6). For 2-propanol, a virucidal activity was observed at a minimal concentration of 30% (vol/vol) with a RF of 5.3.Next, we examined the susceptibility of MPXV against the individual components (ethanol and 2-propanol) of both WHO formulations, which are also the main ingredients of numerous commercially available hand disinfections. Both ethanol , panel AOverall, both WHO formulations efficiently inactivated MPXV, but the 2-propanol‒based WHO formulation II showed a slightly greater virucidal activity than the ethanol-based WHO formulation I. Nonetheless, MPXV showed the greatest stability to both formulations compared with other (re)emerging enveloped or reference viruses.>15 days . A comparative inactivation analyses with different (re)emerging enveloped or reference viruses showed that MPXV had the highest stability against both WHO formulations compared with other enveloped viruses. The susceptibility of the different viruses to the WHO formulations probably depends on virus-specific surface properties of their lipophilic envelope. Nonetheless, our results confirm modified vaccinia Ankara as a suitable model surrogate for MPXV to evaluate chemical disinfectants and antiseptics (Additional information on efficient inactivation of monkeypox virus by World Health Organization‒recommended hand rub formulations and alcohols.


Such interactions triggered shortcomings in the immobilized β-galactosidase (iβGL) thermal and storage stabilities. The thermal stability of the iβGL was somewhat lesser than that of the free βGL. Moreover, the iβGL suffered an initial sharp fall-off in its activity after storing it. Thus, approaches were adopted to prevent the occurrence of such uncontrolled enzyme-immobilizer interactions, and accordingly, boost the stability of the iβGL. These approaches involved neutralizing the covalently reactive GA entities via glycine and also altering the functionalizing GA concentrations. Nonetheless, no improvement was recorded in the iβGL thermal stability and this indicated that the uncontrolled enzyme-immobilizer interactions were not mediated via GA. Another approach was then attempted which involved treating the iβGL with lactose. The lactose-treated iβGL (LT-iβGL) presented superior thermal stability as was verified from its smaller kd and bigger t1/2 and D-values. The LT-iβGL t1/2 values were 5.60 and 3.53 fold higher than those presented by the free βGL at 62 and 65 °C, respectively. Moreover, the LT- iβGL presented loftier ΔG than did the free βGL. The storage stability of the LT- iβGL was also superior as it offered 100.41% of its commencing activity on its 43rd storage day. Thus, it could be concluded that lactose prevented the uncontrolled enzyme-immobilizer interactions. Finally, advantageous galacto-oligosaccharides (GOS) were prepared via the iβGL. The GOS were then analyzed with mass spectrometry, and it was shown that their degree of polymerization reached up to 7.Uncontrolled enzyme-immobilizer interactions were evident after immobilizing  Immobilization is pivotal to render the industrial utilization of enzymes more economically feasible. Immobilized enzymes could be re-utilized many times  and their segregation away from smaller proteins (˂100 kDa) could be fulfilled via immobilization. Bulky proteins would cover more of the immobilizer surface, and accordingly, they could establish distanced multi-interactions. Moreover, they would possess more surface residues that are capable of interacting with the immobilizer  (Wahba Soy-protein isolate (SPI)-GA-functionalized carrageenan (Car) beads were formerly reported as immobilizers for the industrial enzyme L) Wahba . These SL) Wahba . SPI is Galacto-oligosaccharides (GOS) are characteristic for their prebiotic traits as they can promote the propagation of the beneficial microflora within the gastrointestinal tract (Sass and Jördening 2020). Accordingly, GOS are advantageous for humans, and in particular, for babies and infants. Lots of babies consume infant formulas, which are based on bovine milk, as surrogates to the human mothers’ milk, which comprises plenty and diverse oligosaccharides (> 200)  GA were attained from Sigma-Aldrich (Germany). SPI with a protein content of 90% was attained from BulkSupplements.com (USA). Commercial glucose estimation kits were acquired from Spinreact, Spain.Car powder was dissolved in distilled water via stirring in a 70 °C water bath. The hot Car solution  was then extruded through a strait syringe needle into a 3% (w/v) KCl gelling solution. The acquired Car beads were kept in the KCl solution for at least 2 h. Afterward, they were meticulously washed and soaked overnight within 10% (w/w) SPI suspension of pH 5. The beads were then meticulously washed and soaked within GA solution  for 1 h (Wahba 2022a). Finally, the SPI-GA-functionalized Car beads were washed and put in the fridge in distilled water till loading them with βGL.The powdered βGL enzyme was dissolved in citrate–phosphate buffer  and was then mixed with the SPI-GA-functionalized Car beads. Such a mixture was rotated via a roller stirrer for around 18 h. Afterward, the loaded SPI-GA-functionalized Car beads were washed to discard any un-linked βGL entities.The activities of both the free and the iβGLs were estimated. In case of the iβGL, 0.06 g of the βGL-loaded SPI-GA-functionalized Car beads were suspended in 0.5-ml citrate–phosphate buffer . As for the free βGL, it was dissolved in 0.5 ml of the aforementioned buffer. The utilized substrate solution was a 200-mM lactose solution which was prepared in citrate–phosphate buffer . The catalytic reaction was conducted via mixing the 0.5-ml buffer, which contained either the free or the iβGL, with 3.5 ml of the 200-mM lactose solution. The reaction proceeded for 15 min at 37 °C in a shaking water bath. Afterward, a portion of the reaction mixture was removed and was placed in a boiling water bath for around 10 min. After cooling to room temperature, the glucose content of the reaction mixture was evaluated while utilizing commercial glucose kits. One βGL unit (U) indicated that 1 μmol glucose/min was procured while performing the abovementioned catalytic reaction.d) were procured from the slopes. The procured kd values were exploited to derive the half-lives (t1/2) and D-values of the βGL specimens as follows . These specimens were then subjected to thermal incubations at 56–65 °C temperature range. After predetermined durations, the βGL specimens were removed from the thermal incubations and were instantly assayed. The recorded activities were presented relative to the commencing activities which were assessed with no thermal incubation. Plots of log  against time were then constructed, at each inspected temperature, and the first-order thermal denaturation rate constants  was also constructed, and its slope amounted to − 1/z where z was the temperature increment that would induce a tenfold fall-off in the D-values. Furthermore, ln kd values were plotted vs 1/temperature (1/K), and the attained slope corresponded to − Ed/R where Ed was the activation energy of βGL thermal denaturation. The alterations in enthalpy (ΔH), entropy (ΔS), and Gibb’s free energy (ΔG) betwixt the active βGLs and their thermally denatured analogues were also calculated as follows:T was the temperature in K, R was the universal gas constant (8.314 Jmol−1 K−1), h was the Planck constant (11.04 * 10–36 Jmin), and kB was the Boltzmann constant (1.38*10–23 JK−1).Plot of Log A batch of the βGL-loaded SPI-GA-functionalized Car beads was prepared. Some of these beads were assayed directly after finishing the βGL loading process (100% activity), and the rest of the beads were placed in distilled water and kept in fridge. After specific periods, some of the stored beads were assayed, and their activity was presented relative to the 100% commencing activity.In the first approach, the βGL-loaded beads were soaked for 2 h within a 0.2 M glycine solution that was prepared in 0.1 M citrate–phosphate buffer (pH 4.6). The beads were then meticulously washed to remove excess glycine. Afterward, some of these beads were assayed (100%), and the remaining beads were thermally incubated at 56 °C. After predetermined durations, some of the βGL-loaded beads were removed from the thermal incubation and were instantly assayed. The recorded activities were then presented relative to the commencing 100% activity.In the second approach, the βGL-loaded beads were thermally incubated at 56–65 °C temperature range while being soaked in a 200-mM lactose solution. This lactose solution was prepared in 0.1 M citrate–phosphate buffer (pH 4.6). After predetermined durations, the iβGL specimens were removed from the thermal incubation, and the lactose was quickly and meticulously eliminated via washing before estimating the residual iβGL activities. Such residual activities were given relative to the activity of an iβGL specimen which was analogously soaked in lactose but was not thermally incubated.Four approaches were adopted to boost the thermal stability of the iβGL. Two of these approaches utilized the regular βGL-loaded SPI-GA-functionalized Car beads which were prepared and loaded as mentioned above.The two other adopted approaches required modifying the SPI-GA-functionalized Car beads via altering the functionalizing GA concentration from the regular 25–5 or 10%. The 5 and 10% GA-functionalized beads were then loaded with βGL, and their thermal stabilities were estimated at 56 °C.The storage stability of the LT-iβGL was also investigated. Some of the βGL-loaded SPI-GA-functionalized Car beads were initially assayed (100% activity). The rest of the βGL-loaded beads were kept in fridge while being soaked in a 200-mM lactose solution, which was prepared in 0.1 M citrate–phosphate buffer. After specific periods, some of the lactose stored βGL-loaded beads were rigorously washed to eliminate any lactose residues. The beads were then assayed, and the recorded activities were presented relative to the 100% commencing activity.v/v) solvent system was utilized to develop the plates. The locations of the GOS spots were visualized via spraying the plates with a 0.5% 1-naphthol solution, which was solvated in 5% ethanolic sulfuric acid solution, and subsequently heating them. Such locations were marked in analogous unsprayed TLC plats. The unsprayed plates were then put within the Advion plate express, and the GOS spots were extracted via methanol. Afterward, they were analyzed with an Advion compact mass spectrometer with m/z expression range 10–1200. The rate within the spectrometer was put at 0.2 ml/min, and moderate voltage and temperature were adopted . Finally, positive ion electrospray ionization (ESI +) and negative ion electrospray ionization (ESI −) mass charts were acquired.The βGL-loaded SPI-GA-functionalized Car beads were added to a 40% lactose solution so that a ratio of 1.30 U βGL: 1 g lactose was attained. A shaker incubator was then utilized to conduct the βGL transgalactosylation reaction at 50 °C and 100 rpm. After specific durations, samples were collected, and they were suitably diluted with water to spot them on TLC plates. Raffinose, a trisaccharide, was also spotted on the TLC plates. Propanol/water  was also listed in Table S values give an indication of the randomness within a system, and they are usually incremented upon denaturation. Hence, the loftier the ΔS values, the lower the stability of the enzyme  was considered. ΔG is a more dependable enzyme stability indicator as it comprises the enthalpic and the entropic contributions together  remaining epoxy moieties via incubation in glycine , such as isomalto-OS, Xylo-OS, Konjac-OS, or Mycose at a βGL: OS weight ratio of 1:500. The loftiest stability was recorded for the Konjac-OS treated βGL, which kept around 45% of its activity following 1 h incubation at 60 °C  vs 1/temperature. Thus, if the enzyme thermal inactivation became less dependent on temperature, it would be expected that the slope of the plot would attain a lesser absolute value, and accordingly, the Ed (slope =  – Ed/R) would attain a lesser value. This debate could also be applied herein. The LT-iβGL thermal inactivation was less dependent on temperature, and this caused the slope in Fig. Ed was acquired. Analogously, the protease immobilized via an activated gum tragacanth-agar carrier and the carboxymethyl-cellulase immobilized inside agarose presented Ed values which were significantly lower than those presented by their free analogues, and this was also regarded to the fact that the immobilized enzymes thermal inactivation became less temperature dependent . This further verified that the thermal inactivation of the LT-iβGL was less affected and less dependent on temperature. Analogously, immobilizing protease via the activated gum tragacanth-agar raised its z-value from 8.70 to 27.62 °C owing to the lesser dependence of its thermal inactivation on temperature , such as GOS-3, GOS-4, GOS-5, and GOS-6 . Analogously, the high DPN GOS (GOS-6) was formerly reported to be better presented in the ESI- charts than in the ESI + charts (Wahba Thus, the various peaks presented in Fig. ts Wahba .Fig. 9ESG values, which were loftier than those presented by the free βGL at all temperatures. The storage stability of the LT-iβGL was also superior where 100.41 ± 1.08% relative activity was presented on the 43rd storage day. It could also be concluded that the post-immobilization uncontrolled enzyme-immobilizer interactions that occurred amid the iβGL and the SPI-GA-functionalized Car beads were mainly induced via physical interactions mediated via the immobilizer SPI entities.The compromised stability of the SPI-GA-functionalized Car beads iβGL was resolved via simply soaking it with its substrate, lactose. The LT-iβGL presented superior thermal stability as was proved from its thermodynamic parameters especially its Δ