Homo. The GAR IVE hemi-mandible  represents one of the rarely preserved early Homo infants (~ 3 years at death), recovered in a richly documented Oldowan archaeological context. Yet, based on the sole external inspection of its teeth, GAR IVE was diagnosed with a rare genetic disease–amelogenesis imperfecta (AI)–altering enamel. Since it may have impacted the child’s survival, this diagnosis deserves deeper examination. Here, we reassess and refute this diagnosis and all associated interpretations, using an unprecedented multidisciplinary approach combining an in-depth analysis of GAR IVE (synchrotron imaging) and associated fauna. Some of the traits previously considered as diagnostic of AI can be better explained by normal growth or taphonomy, which calls for caution when diagnosing pathologies on fossils. We compare GAR IVE’s dental development to other fossil hominins, and discuss the implications for the emergence of childhood in early Homo.Childhood is an ontogenetic stage unique to the modern human life history pattern. It enables the still dependent infants to achieve an extended rapid brain growth, slow somatic maturation, while benefitting from provisioning, transitional feeding, and protection from other group members. This tipping point in the evolution of human ontogeny likely emerged from early  This provides more time not only for somatic development and protracted brain growth3, but also to learn survival skills4. The offspring is thus dependent upon more parental care over a longer time, and sexual maturity is postponed5. In modern humans, life history stages successively involve infancy, childhood, a juvenile phase, adolescence, and finally adulthood. Different kinds of feeding strategies and dental development stages characterize these phases. Following these criteria, infancy can be subdivided into two stages. First, the “nursing phase” takes place from birth to 6 months, when the infant exclusively relies on breastfeeding and starts erupting its anterior deciduous teeth. Second, the “suckling phase” spans from 6 months to 3 years and involves supplementing complementary food to supply the nutrients not sufficiently present in quantity or quality in the mother milk to enable the fast postnatal brain growth, and the eruption of all deciduous teeth. Weaning is one of the pivotal life history traits occurring during ontogeny: this is a long process involving a transition from exclusive breastfeeding to the progressive introduction of solid food, towards total cessation of mother milk consumption (fully weaned infants) and full reliance on solid diet7, and eventually feeding self-sufficiency. During hominin evolution, major changes in weaning and feeding behaviour may have occurred with early Homo, especially involving the reduction in dental size and dietary shifts to focus on food items more energy dense and easier to masticate, and on an increase in meat and fat consumption and food sharing9. Known as the “Expensive-Tissue Hypothesis”8, this energy-rich diet would have enabled meeting the requirements of this demanding postnatal brain growth and achieving adult brain size by the end of childhood in humans. Following infancy, “childhood” spreads from 3 to 6 years of age when modern human weaned infants show all of their deciduous teeth in functional occlusion, although they cannot yet fully process an adult diet, due to their relatively small size and thin enamel5. In modern humans, the end of childhood is marked by the eruption of the permanent first molar (M1) at ~ 6 years of age1, and the attainment of adult brain size10.The pattern and timing of modern human life history is unique in involving an extended period of growth11. In the tooth microstructure, stress events may manifest as accentuated lines and remain non-specific14. A specific accentuated growth marking called the neonatal line is often identifiable in the enamel and dentine of the deciduous teeth and in the mesial cusps of the permanent first molar which start mineralizing in utero16.In this context, fossil teeth are especially valuable not only because their recovery enables comparing developmental stages between fossil hominins and modern humans but also because dental hard tissues record their own growth and maturation as well as stressful events experienced by the organism18.During both infancy and childhood, protection and food provisioning by adults of the group (parents or others) is crucial for the child’s survival. Understanding the modality and time of emergence of this unique modern life history pattern is currently one of the most debated topics in human evolutionHomo erectus s.l.19, and its age at death was estimated to be 2–3.5 years21. With the recent discovery of H. erectus s.l. remains in association with Acheulean and Oldowan artifacts at Gona 22, GAR IVE is one of the rare early Homo remains recovered from the beginning of the Early Pleistocene, in the context of proper archaeological excavations providing contextual information on hominin activities23. GAR IVE was found with Oldowan industry and faunal remains in layer E, later dated to ~1.8 Ma  is a fragmentary right hemimandible with a partial mixed dentition, recovered in layer E at the Garba IV site of the Melka Kunture complex  19, although they were only based on classical radiographs and SEM imaging of epoxy replicas of the tooth surface30. Zanolli and colleagues worked on CT data, but did not re-evaluate this diagnosis21. Amelogenesis imperfecta encompasses a group of developmental conditions31 altering the enamel structure and making teeth fragile32. The main diagnostic criteria may involve a marked reduction in enamel thickness frequently concerning both deciduous and permanent teeth, enamel pits or linear hypoplasia not associated with any noticeable developmental defects in the other dental tissues, a brown colouration of the enamel, or taurodontism 36. In some AI variants, enamel defects may be associated with other types of syndromes occurring in other parts of the human body 38. In clinics, a differential diagnosis requires the knowledge of medical and family history of the patients, of their genetic profile, as well as of their conditions of development and life. Thus, AI patients mostly suffer from early tooth loss, severe embarrassment, eating difficulties and pain, requiring early and complex restorative treatments38.Previous studies reported GAR IVE as being affected by a rare genetic disease 38. According to Zilberman et al.30, the presence of AI in GAR IVE would provide evidence of genetic continuity from early Homo to modern humans. Yet, this claim has to be considered with caution since the molecular mechanisms causing this pathology are complex and may involve several genes and combinations of genes39. Such a genetic continuity is unlikely to be so straightforward. Furthermore, Trinkaus40 has suggested that any variant of this pathology would have involved the need for enhanced maternal care to supply the young child with sufficient nutrients and permit its survival to some extent40.Genetic diseases are of particular interest as they happen to be transmitted along a lineage affecting multiple generations, and they may occur with variable frequencies, in the case of AI, ranging from 1:700 to 1:14 000Homo infant.Given the putative consequences of this disease on the specimen’s morphology and the potential implications of living with AI during the Pleistocene, we aim to reassess the diagnosis of AI by analyzing tooth surface features and by exploring the fine internal structure of the bone and teeth using synchrotron imaging. We compare the GAR IVE teeth with teeth from modern human patients, some with documented forms of AI. Because taphonomic processes may affect the preservation of the specimens and the features related to the AI diagnosis, our study includes the analysis of the associated faunal remains and the geological context in which GAR IVE was recovered in order to establish if particular features are due to taphonomy or to developmental alterations induced by the AI. In spite of the taphonomic damages undergone by the dental tissues, we provide some novel fragmentary yet still valuable palaeobiological data on the dental development of this early After a brief description of the GAR IVE specimen, and especially of its dental development, we have compared the enamel features described as diagnostic of AI on GAR IVE to the enamel condition in teeth of modern-day AI patients. Then, we have explored the fine microstructure of the teeth and bone in light of taphonomic processes, recovered from the study of associated faunal remains, and the palaeoenvironmental reconstruction of the fossilisation context of GAR IVE.1 and LRdm2). The LRdm1 is considerably worn (yet preserving a complete rim of enamel) and super-erupted above the occlusal level. The germ of the permanent first molar (LRM1) is partially exposed at the posterior part of the specimen, as the mandibular ramus is missing. Its crown does not present any abnormal morphological features; to note that its occlusal surface shows extensive wrinkling, yet this LRM1 looks similar to that of other fossil hominins  visible and partially unprotected by its bony crypt. The germs of the permanent canine (LRC1), third and fourth premolars (LRP3 and LRP4) are unerupted and protected inside the mandibular bone. The bone and teeth present various kinds of surface alterations.The GAR IVE right hemimandible Fig.  preserveins Fig. . The ant42, the overall calcification stages in GAR IVE would correspond to 4.5 years of age  seems to be developmentally the closest to GAR IVE and has an estimated age at death of 2.5–3.0 years. KNM-ER 1820 (P. boisei) is slightly more advanced than GAR IVE for an age at death similar to KNM-ER 812 (2.5–3.1 years). Overall, the calcification of the GAR IVE permanent tooth germs is less advanced than that of Sts 24 .The synchrotron µCT scans show a loss of contrast between bone, enamel and dentine, the enamel-dentine junction being barely visible in some places Fig. . Any est44 show that they occurred every ~ 1.4 months, from 2.5 to 2.9 years of age (see Supplementary Text Homo) LRP4 matches with the purple stress “S3” in GAR IVE . Death occurred ~ 77 days later in GAR IVE, which correspond to ~ 873 days or ~ 2.4 years in the DNH 35 growth pattern. “S3” shows that the calculated estimation is slightly ahead of the developmental stage observed in other early Homo specimens (here by 6.5 months at most), yet this is may be accounted for by the variability which is not captured here, and by the uncertainties related to our measurements .In spite of the taphonomic alteration of the dental tissues in GAR IVE, the synchrotron data revealed several accentuated lines in the LRP4  are distributed close to each other on the anterior portion of the mandibular corpus  can also be observed on the teeth and bone of GAR IVE Fig. . They dine Figs. , 7. Thesace Fig. . TherefoOn the lingual side of the mandibular corpus of GAR IVE, four striae point to teeth sharpening by a small rodent , as well as reduced enamel radio-opacity19. In agreement with Zilberman et al.19, Trinkaus40 further concluded that GAR IVE had suffered from restricted functional masticatory capacity resulting from AI, which induced malnutrition around weaning age.Reaching a palaeopathological diagnostic on fossil remains often reveals challenging, even more so as the latter ones are usually fragmentary and damaged. Yet, this has always been attempted, ever since the nineteenth century, with mixed results, e.g., the Neanderthal specimen from Feldhofer Grotte19 ‘s diagnostic, GAR IVE would most likely have been affected by the AI variant called “hypoplastic, pitted autosomal dominant” Type IA as defined by Witkop Jr.33. In this variant, enamel may be totally absent or often fragile and significantly thinner to the point of breaking and eventually being totally lost61. The enamel surface is often rough, smooth or pitted. This is not the condition observed in GAR IVE: neither preserved deciduous nor permanent teeth are brittle or crumble, partly due to their normal enamel thickness . This supports the hypothesis that this trait is not pathological and rather results from the retention of a primitive morphology in healthy modern humans, and in GAR IVE.The extensive wrinkling of the occlusal surface of the GAR IVE LRM1 Fig.  looks siRM2 Fig.  resemble1, no comparable dental defects could be found in the medical literature. Towle and Irish71 describe that Paranthropus robustus molars show the most frequent occurrence of pitting enamel hypoplasia (PEH) than any other extant Primates studied. They further notice that the pits are more frequent on deciduous than on permanent teeth, the anterior teeth not being affected. Towle and Irish71 make a correlation between this PEH and the pits observed in modern humans affected by AI, and thus propose that these pits have a genetic origin. If the defects observed on GAR IVE had a genetic and developmental origin, their edges—which were shown to be sharp  showed that this juvenile, deceased at 8–9 years of age, presented an advanced skeletal and dental maturation suggesting that growth was rapid, with the lack of a slowdown growth characteristic of childhood11. KNM-ER 1507 and KNM-ER 820 . The comparison of GAR IVE with modern human equivalents as described by AlQahtani and colleagues42 show a tremendous variation in mosaic among tooth type  has the permanent first molar in occlusion and already worn74 confirming a rapid growth in this species. Early Homo at around 1.5–1.8 Ma would not have experienced any significant period of slow somatic growth between the end of infancy and the juvenile stage76. Thompson and Nelson posit that childhood bears a cost to the group of hominins5. Although having a stage of childhood decreases child mortality and increase the fertility rate of the mothers, the pressure increases on the whole group to care for the offspring in terms of provisioning and protection during their least productive years of life5. Since taphonomy has obscured most the dental microstructure in the GAR IVE teeth, a fine quantitative documentation of the pattern and timing of its dental development remains yet inaccessible. The accentuated lines occurring in some of its tooth germs could have been imprinted by strong stress events occurring before death. It remains however totally speculative to propose that these repeated stress events may have had an impact on the chances of survival of the child, or its health and growth. The circumstances of the death of GAR IVE at a very early stage of its life – presumably before 3 years of age – remains unelucidated. However, our findings proposing that GAR IVE has not suffered from AI restore the status of this early Homo infant as not being affected by a pathology which could have been lethal. The tooth morphology of GAR IVE can thus be reliably included in future comparative studies, such as for refining its taxonomic status.Whether the ontogenetic stage “childhood” has emerged from the 30, several cannot be grounded in GAR IVE. We demonstrated that some features interpreted as symptoms of AI are normal anatomical features related to bone growth  and tooth eruption , while others are resulting from post-mortem process. In general, there is no evidence of a pathological enamel condition in GAR IVE, and all these observations invalidate the diagnosis of AI in this early Homo juvenile. GAR IVE can thus be reliably used in future comparative studies as one of the rare early Homo infant specimens preserving a partial mixed dentition.High-resolution synchrotron imaging allowed reassessing the diagnosis of AI in GAR IVE. Among the arguments raised by Zilberman and colleaguesOverall, our results suggest caution when diagnosing a rare genetic disease on a fossil remain, and considering its implications on our understanding of hominin life history and consequently, on human evolution. Fossil human remains gain to be investigated using an integrative approach, combining different fields of expertise, such as taphonomy, environmental reconstruction, archaeological and geological context, high-resolution imaging techniques and/or a clinical approach, whenever needed.The GAR IVE mandible is kept in the National Museum of Addis Ababa (Ethiopia) together with the archaeological and palaeontological collections from Garba IV and all other sites of Melka Kunture. ARCCH  provided the permit for the temporary export of the fossil to be scanned in Grenoble (France) at the European Synchrotron ESRF.11. In the enamel, stress events may manifest as accentuated lines which remain non-specific14. To note that birth is recorded by the neonatal line often identifiable in the enamel and dentine of in deciduous teeth and the mesial cusps of the permanent first molar which start mineralizing in utero16.Since the variously-sized and -shaped surface morphology on the GAR IVE teeth had been interpreted as abnormal and lead to reach the general diagnosis of AI, we explored the tooth surface features. Concerning the developmental alterations that AI produces, the analysis of the enamel microstructure is especially valuable. Indeed, dental hard tissues record their own growth and maturation as well as stressful events experienced by the organism77.To access the tooth and bone microstructure of GAR IVE, we scanned the mandible and its dentition at high-resolution using synchrotron propagation phase contrast micro-computed tomography (PPC-SR-µCT). The scans were acquired by Dr. Paul Tafforeau at the ESRF  on the ID 19 beamline. The technical parameters used for the data acquisition are listed in detail in Supplementary Text To reassess the diagnosis of AI in GAR IVE, and further study the 3D enamel thickness distribution in its LRM1, we aimed to access clinical extractions of teeth from patients diagnosed with AI. Despite a wide query in clinical institutions in several countries, we could eventually only access three teeth. The first tooth  in Fig. 78. The taphonomic observations conducted on GAR IVE and the associated faunal remains were performed using 10 ×-20 × hand lenses and a Dino-Lite Edge 5MP, at the National Museum, in Addis Ababa, Ethiopia.Faunal remains were examined to determine what taphonomic process have been a play at the site, and whether they could have produced the features observed on GAR IVE  and interpreted as being related to the AI pathology. Taphonomy encompasses the characteristics of the general environment during fossilization. Our approach involved the reconstruction of the palaeonvironmental context of Garba IVE, which is based on the detailed analysis of the stratigraphic sections carried out in 2013 and 2017. Sedimentary features at the meso- and micro-scale  were studied for the reconstruction of the depositional processes, volcanic facies and for correlation with the stratigraphic sections along the Garba and Gombore gully. The results were eventually correlated with the dataset of Raynal and KiefferSupplementary Information.


Chondroblastoma is an uncommon osseous neoplasm that accounts for less than 1% of all bone tumors. Characteristically it arises in the epiphysis or apophysis of long bones in young patients and may extend into the metaphysis. A sixteen-year-old male presents with a 1-year history of left knee pain associated with joint stiffness and interfering with performing daily activities. Radiographic and contrast enhanced magnetic resonance imaging favored the diagnosis of chondroblastoma. This was then confirmed histologically. The clinical signs and symptoms of Chondroblastoma are usually nonspecific, pain is most often moderate and can be revealed as a result of a trauma. The definitive diagnosis is mainly pathological due to the lack of specificity of radiological findings consistent with the presented case. Chondroblastoma presented a puzzling classification conundrum for early physicians. First remarked on by Ewing as a “calcifying” giant cell tumor associated with cartilage resorption generally at the humeral head and then by Codman in his review of nine cases from the Bone Sarcoma Registry as “epiphyseal chondromatous” giant cell tumors A sixteen-year-old male presents with a 1-year history of left knee pain associated with joint stiffness and interfering with performing daily activities. Radiographic and contrast enhanced magnetic resonance imaging favored the diagnosis of chondroblastoma. This was then confirmed histologically.The patient is a basketball player, however since onset of the knee pain, he has not played and avoids physical activity. Physical examination of the left knee reveals diffuse tenderness and moderate sized joint effusion.  There is no fever or any other sign of systemic illness and laboratory findings were normal. Plain radiographs exhibit a lucent rounded lesion with sharp sclerotic margin within the distal anterior femoral metaphysis and epiphysis. . MRI of Chondroblastoma is a rare type of benign cartilage tumor that accounts for approximately 1% of all bone tumors. It most frequently affects the skeletally immature so it is mostly encountered in children and young adults between ages 19 to 23 with a M>F ratio of 2:1 [7]Generally, the presentation is nonspecific with common presenting symptoms including mild localized pain, swelling, and loss of range of motion of the affected joint. This is thought to be the result of chondroblastoma's production of prostaglandins Typically, on radiograph chondroblastoma presents with an eccentrically or centrally located osteolytic lesion with either smooth or lobulated margins and thin sclerotic rim involving the epiphysis or other secondary ossification centers. Chondroblastoma is nonprogressive if left in situ. Treatment depends on the anatomic location of the lesion and the extent of bone and/or joint involvement. It is usually treated by curettage and bone graft. Chondroblastoma is a rare type of benign cartilage tumor that accounts for approximately 1% of all bone tumors. It most frequently affects children and young adults. The clinical signs and symptoms are usually nonspecific, pain is most often moderate and can be revealed as a result of a trauma. The definitive diagnosis is mainly pathological due to the lack of specificity of radiological findings consistent with the presented case. The treatment is almost always surgical curettage and bone grafting. The functional prognosis of chondroblastoma depends on its location and its degree of aggressiveness as defined by joint destruction and nearby bony extension.


At the same time, MET improved motor function recovery in rats after SCI by reducing motor neuron loss in the anterior horn of the spinal cord. Taken together, our study confirmed that MET inhibits neuronal pyroptosis after SCI via the AMPK/NLRP3 signaling pathway, which is mostly dependent on the AMPK pathway increase, hence decreasing NLRP3 inflammasome activation.Spinal cord injury (SCI) is an extreme neurological impairment with few effective drug treatments. Pyroptosis is a recently found and proven type of programmed cell death that is characterized by a reliance on inflammatory caspases and the release of a large number of proinflammatory chemicals. Pyroptosis differs from other cell death mechanisms such as apoptosis and necrosis in terms of morphological traits, incidence, and regulatory mechanism. Pyroptosis is widely involved in the occurrence and development of SCI. In-depth research on pyroptosis will help researchers better understand its involvement in the onset, progression, and prognosis of SCI, as well as provide new therapeutic prevention and treatment options. Herein, we investigated the role of AMPK-mediated activation of the NLRP3 inflammasome in the neuroprotection of MET-regulated pyroptosis. We found that MET treatment reduced NLRP3 inflammasome activation by activating phosphorylated AMPK and reduced proinflammatory cytokine (IL-1 External direct or indirect sources are the most common causes of spinal cord injury (SCI), resulting in a permanent disability below the level of injury . PrimaryMetformin (MET) is a hypoglycemic drug commonly utilized in the treatment of type 2 diabetes , as it gAs a newly discovered process of programmed cell death, pyroptosis has received increasingly attention to researchers, and it often takes place to various stress environments . PyroptoThe capacity of MET to protect neurons from acute SCI damage was studied in this study, as well as whether the protective mechanism of MET involves the modulation of the AMPK/NLRP3 pathway.2. The cells were haphazardly partitioned into 4 groups: (1) control group: no additional treatment was added to the cell culture mediums, (2) lipopolysaccharide (LPS) group: 100 ng/mL LPS was added to the cell culture mediums for stimulation for 24 h, (3) MET group: 50 μM of MET was added to the culture medium after the treatment with LPS, and (4) MET+compound C (CC) group: 50 μM of MET and 50 μM of CC were added after the treatment with LPS. The selection of drug dosage was based on previous studies [Primary neurons were removed from the spinal cord of newborn (<48 h) rat pups, the neonatal mouse pups were euthanized by rapid neck-breaking, and the tissues were minced and gathered and afterward processed with EDTA containing 0.25% trypsin for 15 min at 37°C. After the absorption was ended, the cells were cultivated onto poly-DL-lysine-coated glass coverslips  in 24-well plates and refined in Dulbecco's modified Eagle's medium (DMEM) containing 10% foetal bovine serum (FBS) and 25 mg/ml penicillin/streptomycin . RAW 264.7 cells (procured from the American Type Culture Collection (ATCC)) were refined in DMEM medium containing 10% FBS. All cells were grown at 37°C in 5% CO studies .μM). Then, at that point, after 24 h hatching, MTT  was added to each well, and the plates were kept under the right conditions for growth for 4 h at 37°C. Next, 150 μL dimethyl sulfoxide  was added for 10 min at 37°C to dissolve the crystals. Finally, a microplate peruser was utilized to gauge and record the absorbance at 490 nm.The decrease of 3--2,5-diphenyltetrazolium bromide (MTT) to a purple formazan item was measured to decide the impact of MET on cell survival. In short, after LPS induction, the cells were treated with MET , anti-GAPDH , HRP Affinipure Goat Anti-Mouse IgG , and HRP Affinipure Goat Anti-Rabbit IgG . The outcomes were investigated with an UVP Electrophoresis Gel Imaging System (US) and ImageJ programming.Western blot methods were performed according to previously studied . The accβ-Tubulin  followed by washes with PBS and incubation with an Alexa Fluor®488-conjugated goat anti-mouse/rabbit IgG antibody for 2 h at room temperature. 4,6-Diamidino-2-phenylindole  was used to stain nuclei. A high-resolution confocal microscope was used to observe the cells  and examined by the ImageJ software.Primary neurons and RAW 264.7 cells filled in a confocal petri dish were washed with super cold PBS  and fixed with super cold 4% paraformaldehyde for 30 min. After the cells were washed with PBS for multiple times, they were impeded with typical goat serum containing 0.1% Triton X-100 for 2 h at 4°C. The cells were then incubated overnight at 4°C with the appropriate anti-NLRP3 , anti-p-AMPK , anti-NeuN , or anti-β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in cell supernatant or serum concentration. The operation method of the ELISA kit was in accordance with the instruction of the instruction manual.The enzyme-linked immunosorbent assay (ELISA) kit  was utilized to recognize the inflammatory cytokines interleukin-1Sprague-Dawley (SD) female rats  were bought from Liaoning Changsheng Biotechnology Co., Ltd. . All experimental procedures complied with the Care and Use of Laboratory Animals guidelines published by the US National Institutes of Health and were approved by the Laboratory Animal Ethics Committee of Jinzhou Medical University. Animals were reared at a temperature 23 ± 0.5°C with day and night light to simulate the natural environment. The test creatures were partitioned into 3 gatherings similarly: the Sham group, which underwent laminectomy only, the Vehicle group, which went through a medical procedure for SCI and got Vehicle  right away and 24 hours after surgery, and the MET group, which went through a medical procedure for SCI and got MET  right away and 24 hours after surgery. The portions of MET administered depended on past studies [Anaesthetized  rats were put in a prone position on a cork platform. There was no rat displayed indications of peritonitis, torment, or inconvenience following i.p. administration of sodium pentobarbital. The surgery was performed under sterile conditions, and the T9/10 lamina was excised after incision of the skin. The SCI model was built using Allen's technique . The impIn an open field, the Basso, Beattie, and Bresnahan (BBB) scores and the inclined plane test were administered on days 0, 1, 3, 7, 14, 21, and 28 after SCI to assess motor recovery in all animals. All tests were performed by specialists double-blinded to the treatments.μm transverse cryosections). After HE staining, histological alterations at the injury site were evaluated, and the number of anterior horn motor neurons was counted.After 28 days, the rats were perfused with 0.9% saline and 4% paraformaldehyde, and the spinal cord tissue was extracted. The spinal line fragments, including the injury site, were cut utilizing a cryostat  , endogenous peroxidase action and vague antigens were hindered with 5% typical bovine serum. The segment was then treated overnight at 4°C with anti-NLRP3 . The primary antibody was not used in the negative controls. After then, a biotinylated anti-rabbit IgG antibody was used to bind the section, which was subsequently followed by HRP-conjugated streptavidin. A biological imaging microscope was used to capture the images. The result of immunohistochemical analysis is the optical density value of the picture , namely, the OD value. The deeper the immunohistochemical DAB staining, the greater the OD value and the more protein expression. The Image J software was used to determine the OD value obtained by DAB staining, and the outcomes were analyzed utilizing the SPSS 19.0 software.P values < 0.05 were considered as genuinely critical.The mean ± standard error of the mean (SEM) was used to communicate the data, which was broken down using SPSS 19.0 programming. To make a correlation of the differences across different gatherings, one-way analysis of variance (ANOVA) or two-way inquiry of fluctuation was used, followed by Bonferroni's post hoc test. μM MET treatment ; therefore, a dose of 50 μM was selected for all subsequent experiments.The MTT assay was performed to determine cell viability after cells were treated with MET  for 24 hours. Rat neuronal cell  and RAW in vitro, MET and CC were added in the cells. Then, the levels of p-AMPK, AMPK, p-ACC, ACC, NLRP3, ASC, Caspase 1, and IL-1β were performed by Western blot analysis. As shown in ∗∗∗P < 0.001, n = 3).Primary neurons were treated with LPS to establish a model of injury. To demonstrate the effect of MET on the AMPK/NLRP3 signaling pathway ∗∗∗P < 0.01, n = 3). In addition, immunofluorescence showed that the levels of p-AMPK were significantly inhibited by MET in LPS-treated primary spinal cord neurons, and CC reversed this phenomenon . These findings imply that MET activates the AMPK/NLRP3 signaling pathway and suppresses pyroptosis.The expression of NLRP3, ASC, Caspase 1, and IL-1 was considerably downregulated after LPS-induced cells were treated with MET . The MET group's inflammation level was lower than the LPS group's, whereas the MET+CC group's inflammation level was higher than the MET group's. We concluded that MET significantly prevented LPS-induced upregulation of inflammatory factor levels. This could be linked to MET's regulation of NLRP3 expression.To explore the ability of MET to inhibit inflammation, RAW 264.7 cells treated with LPS were used as an inflammation model ∗P < 0.05, n = 3). The results of the inclined plane test were similar to the BBB scores . These findings indicated that MET can improve motor function following SCI.The BBB scores served as a proxy for the beneficial effect of MET on locomotor recovery following SCI. The BBB score of the Sham group was normal, and there was no measurable difference between the MET group and the Vehicle group at 1 and 3 days after injury. Regardless, the scores in the MET group were higher 7 days after SCI . These results indicated that MET was able to reduce motor neuron death after SCI.On day 28 after SCI, HE staining revealed changes in the neurons of the ventral horn of the spinal cord. The number of ventral horn motor neurons was considerably larger in the MET group than in the Vehicle group . Next, we examined whether MET could reduce pyroptosis in vivo by activating AMPK phosphorylation. As shown in Figures β and a significant decrease in MET group (∗∗∗P < 0.001). MET inhibited SCI-induced activation of NLRP3 complex. Furthermore, immunohistochemistry was utilized to distinguish the levels of NLRP3 protein after SCI, which were importantly dropped in the MET treatment group compared with the levels in the other groups . These outcomes showed that MET could activate the AMPK/NLRP3 signaling pathway and reduce pyroptosis after SCI.To determine whether the AMPK pathway is taken part with the effects of MET treatment after SCI, Western blot was used to look for the levels of p-AMPK, AMPK, p-ACC, and ACC. The MET treatment group had significantly increasing levels of p-AMPK and p-ACC than the Vehicle group . In comparison to the Vehicle group, the MET group had lower levels of inflammatory factors . These findings indicated that MET inhibited the release of proinflammatory factors after SCI.The effect of MET on the release of inflammation-related cytokines after SCI was investigated in this experiment. In the spinal cord tissue homogenates, ELISA measured the amounts of IL-1in vivo and in vitro by inhibiting nerve cell pyroptosis and inflammation. MET is a biguanide hypoglycaemic agent normally used to treat type 2 diabetes [As far as we have knowledge of, this is the first learning process to investigate the regulation of AMPK/NLRP3 inflammasome pathway by MET after SCI. We found that MET has a neuroprotective effect diabetes . A largediabetes , 8, 11. diabetes , 23, 24.diabetes , 26.β and IL-18, which will stand out from more provocative cells and aggravate the inflammatory response. The inflammasome is a multiprotein complex that plays a direct role in the inflammatory process by causing Caspase 1, IL-1, and IL-18 to become overactive. Inflammation can worsen tissue necrosis and apoptosis following SCI, according to studies, and the NLRP3 inflammasome plays a key role in this [“Pyroptosis, also experienced as cell inflammatory necrosis, is a new way of programmed cell death discovered and confirmed in recent years” , 27. It  in this , 34. NLR in this . NLRP3 i in this , 36.Ischemia and hypoxia, immunological inflammation, free radical damage, and lipid peroxidation are all pathogenic events in secondary SCI . Preventin vitro. We found that MET treatment activated AMPK and its downstream protein ACC and restrained NLRP3 inflammasome enactment in primary neurons. The AMPK inhibitor CC reversed the suppression of pyroptosis-related proteins caused by MET therapy. At the same time, macrophages/microglias are effector cells of the immune response in CNS [β, IL-6, and TNF-α was shown to be considerably suppressed by MET. These findings suggest that MET has a powerful anti-inflammatory impact, which is associated with actuation of AMPK and restraint of NLRPR initiation.In this study, primary neurons and RAW 264.7 cells were used to simulate the SCI model e in CNS , so RAW in vivo. We established SCI model of rats. We found that MET treatment reduced neuronal loss and improved motor function recovery after SCI. At the same time, MET treatment activated the spinal cord tissue AMPK signaling pathway and inhibited NLRP3 inflammasome activation. ELISA results also confirmed that MET inhibited the expression of inflammatory factors. These results are consistent with those of in vitro experiments.To further verify the mechanism of action of MET The molecular mechanism of MET treatment of SCI is complex, involving multiple molecular pathways. Our study showed that MET inhibited pyroptosis and inhibited inflammatory responses after SCI by activating AMPK and its regulated NLRP3 inflammasome. Therefore, the regulation of NLRP3 by AMPK may give another helpful idea and strategy for the treatment of SCI.Ultimately, this research is the first to establish that metformin hinders neuronal pyroptosis and works on utilitarian recuperation after SCI through the AMPK/NLRP3 signaling pathway. Our outcomes suggest that metformin might serve as a therapeutic intervention to reduce inflammation and the effects of acute SCI.


Given the overlapping clinical features of sino‐nasal immunoglobulin 4‐related disease (IgG4‐RD) to rhinitis or rhinosinusitis, this paper aims to delineate this rare, isolated manifestation significant to physicians for their daily practice and researchers contributing to this field. Given the overlapping clinical features of sino‐nasal immunoglobulin 4‐related disease (IgG4‐RD) to rhinitis or rhinosinusitis, this paper aims to delineate this rare, isolated manifestation significant to physicians for their daily practice and researchers contributing to this field. After urgently ruling out common malignant sino‐nasal tumors like squamous cell carcinoma and lymphoma, this disease must be high in differential not to delay diagnosis and treatment. Currently, no definitive diagnostic criteria for IgG4‐RD affecting the sino‐nasal cavity are established; rather, a 3‐tiered diagnostic algorithm in a consensus statement was proposed during the first international symposium on IgG4‐RD in 2011.Symptoms mimicking rhinitis or rhinosinusitis including facial pain due to pressure, bloody nasal discharge, and nasal obstruction may prompt prescription of steroid sprays in the clinic, possibly delaying or missing investigating for IgG4‐RSD, which is only an emerging disease in the literature.5We hope this case report promoted broader recognition of IgG4‐RD among physicians and researchers, also highlighting the extremely rare manifestation of IgG4‐RD in the sino‐nasal cavity. While ruling out malignancy, lymphoma and infection are critical before considering IgG4‐RD, and investigations such as histopathology and serology should not be delayed maximizing patient outcome given studies have shown good prognosis with early intervention. In terms of demographic, it would be interesting to learn more about patient cases from different parts of Asia who may contribute to this reported predisposition for head and neck manifestations of IgG4‐RD.None declared.HAS and MZS: involved in manuscript writing. KS, AAA, and AS: involved in data collection and manuscript writing.Consent obtained from the patient is available upon request.


Polyhedral oligomeric silsesquioxanes (POSS) have attracted considerable attention in the design of novel organic-inorganic hybrid materials with high performance capabilities. Features such as their well-defined nanoscale structure, chemical tunability, and biocompatibility make POSS an ideal building block to fabricate hybrid materials for biomedical applications. This review highlights recent advances in the application of POSS-based hybrid materials, with particular emphasis on drug delivery, photodynamic therapy and bioimaging. The design and synthesis of POSS-based materials is described, along with the current methods for controlling their chemical functionalization for biomedical applications. We summarize the advantages of using POSS for several drug delivery applications. We also describe the current progress on using POSS-based materials to improve photodynamic therapies. The use of POSS for delivery of contrast agents or as a passivating agent for nanoprobes is also summarized. We envision that POSS-based hybrid materials have great potential for a variety of biomedical applications including drug delivery, photodynamic therapy and bioimaging. Hybrid materials have attracted tremendous attention recently due to their potential applications in several fields such as energy, biomedicine, materials, catalysis, and others ,2,3,4. H1.5)n , where R represents either a hydrogen atom or organic groups. The most common form of POSS is n = 8, also known as a POSS cage. In this review, we will use the words POSS cage and POSS interchangeably, unless otherwise specified. POSS molecules consist of a core cubic cage of eight silicon corner atoms and twelve oxygen edge atoms (Si-O-Si), where each of the silicon atoms may carry an organic group surrounding the periphery of the cage. These groups can be further modified to afford hundreds of possible compounds. POSS combine many of the benefits of silica  with those of siloxane polymers , as well as the potential to further modify their structure by functionalizing R to achieve the desired properties  is a class of hybrid materials with unique 3D configuration, definite framework, and customizable physicochemical properties. POSS may be considered as the smallest possible form of hybrid silica-based materials with sizes of 1–3 nm ,8. The boperties . All theIn the past decades, POSS have been used as building blocks, crosslinkers, and nanofillers to afford hybrid materials with improved stability, mechanical properties and chemical resistance . POSS haPOSS typically have rigid 3D nanosized structures with a diameter of approximately 1–3 nm, which provides several advantages to POSS monomers in building sophisticated hybrid materials . In factThe functionalization of POSS molecules can render endless possibilities to engineering materials with a desired performance. A wide variety of functional groups, such as olefins, acrylates, phenols, fluoroalkyls, halides, amines, sulfhydryls, azides, nitriles, phosphines, carbazoles, imidazolium salts, azobenzenes, cinnamates, etc., can be used for this purpose ,7. Some Depending on the molecular design requirements and reactivity of corner substituents, POSS could incorporate into a polymer matrix as grafted chains or act as cross-linking sites. The recent development of controlled/living radical polymerization techniques, such as atom transfer radical polymerization (ATRP), reversible addition-fragmentation chain transfer polymerization (RAFT), ring-opening metathesis polymerization (ROMP), and anionic polymerization allow for a better control of the polymerization process. The accessibility to different functional groups, in combination with click chemistry, has allowed researchers to prepare reactive POSS more efficiently to develop high-performance hybrid materials. These polymerization strategies have been used to fabricate polymer POSS with different architectures, including tadpole-, dumbbell-, multi-telechelic, star-like, and dendritic ,4,13. In recent years, the use of building blocks that can be precisely designed to fabricate hybrid materials through self-assembly has been a burgeoning field of research. The hydrophobicity and low surface energy of POSS provides a strong tendency to aggregate, which contributes to the controlled self-assembly and confined motion of polymer chains. In turn, these properties lead to the desired topologies and physicochemical properties ,24. In a3 or RSi(OR’)3 precursors 3 precursors, many of which are commercially available. This reaction generally affords good yields and POSS molecules with a wide variety of functional groups such as amino, thiol, vinyl, halogen, etc. For instance, our group used this strategy to afford aminopropyl-hepta(isobutyl)- or aminopropyl-hepta(phenyl)-POSS from the corresponding trisilanol POSS precursors. These molecules were further modified to finally obtain porphyrin-POSS molecules . PO. PO8) vi3 and N,N-dimethylformamide (DMF) . Th. Th55]. 1O2) is generated in response to lysosomal acidic conditions, and efficient lysosomal escape occurs via the photochemical disruption of lysosomes. This team has incubated A549 cells with free HCPT or PPR/HCPT NPs at different incubation periods to investigate their cell internalization capacity to the cancer cells and several analytical techniques to observe the consequences after nuclear entry. They concluded that the released nanoparticles could accumulate on nuclear membranes and augment membrane permeability by inducing lipid peroxidation upon further light exposure.Nuclear membrane and nuclear pore complexes (NPCs) are a barrier for nucleus targeting drugs like 10-hydroxycamptothecin (HCPT) and docetaxel (DTX). Fu-Gen et al., offer a platform that consists of a polyamine-containing polyhedral oligomeric silsesquioxane (POSS) unit, a hydrophilic polyethylene glycol (PEG) chain, and the photosensitizer rose bengal (RB) that can self-assemble into nanoparticles (denoted as PPR NPs)  98]. Wh. Wh98]. Fu-Gen and group members have crosslinked a carboxyl-containing photosensitizer, Chlorin e6, into an amine-containing POSS structure. An amine-carboxyl reaction forms a 3D network via 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide (EDC)/N-hydroxysuccinimide (NHS) coupling method. The resulting POSS-Ce6 shows an improved dispersibility in aqueous media and prolonged circulation time after PEG coating. The final nano agent (POSS-Ce6-PEG) provides a high degree of chemical stability in PDT with significant drug-loading capacity .Lee and Kim conjugated Protoporphyrin IX (PpIX) molecules and linolenic acid to a water soluble octammonium POSS. The molecules self-assembled into nanoparticles of ~120 nm in diameter due to strong hydrophobic interactions . CellulaZhang and co-workers synthesized a unimolecular micelle based on an amphiphilic star shaped block copolymer, which is made of hydrophobic poly(E-caprolactone)(PCL) and hydrophilic poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA). The photosensitizer pheophorbide A was loaded in the hydrophobic portion in the inner layer of the micelle . AdditioJin and co-workers incorporated pendant isobutyl POSS monomers into an amphiphilic copolymer that alternates a tetraphenyl porphyrin with the POSS. With this arrangement, the authors prevented porphyrin aggregation and reduced the AIQE . The sysOur group has also functionalized POSS molecules containing different substituents linked to a porphyrin photosensitizer . A serieE. coli) due to its high positive charge density. Moreover, under white light irradiation, the POSS-porphyrin platform eliminated 99.9% of Gram-negative E. coli or Gram-positive S. aureus using 8 μM or 500 nM, respectively  where the POSS arms were functionalized with PEG5000 to create water-soluble nanoparticles with a hydrodynamic diameter of 28 nm  49]. Th. Th49]. Wu and co-workers used octaaminopropyl POSS as a building block to fabricate a nanoparticle which contains a network of crosslinked Ce6 photosensitizers . The resZhang and co-workers utilized POSS as a template for the fabrication of hollow nanoparticles. The system was synthesized using an amphiphilic block copolymer containing the light/redox-responsive monomer coumarin methacrylate, the pH-responsive hydrophilic monomer 2-(dimethyl amino) methacrylate, and an isobutyl POSS based methacrylate monomer . This poIn recent decades, bioimaging has attracted much attention due to its ability to obtain anatomical and physiological details of bio-systems ranging from cell to ex vivo tissue samples and in vivo imaging of living objects . BioimagRotello and coworkers pioneered the use of POSS for bioimaging applications. They reported on the modification of octaammonium-POSS (OA-POSS) with boron-dipyrromethene (BODIPY) as a cellular fluorescent marker . The POS1H and 29Si NMR, and mass spectrometry. The light absorption properties of POSS_F are similar to the parent fluorophore. However, the emission intensity of POSS_F increased approximately four times compared to the corresponding equimolar FITC solution. The authors evaluated the in vitro performance of POSS_F using fluorescence and confocal microscopy in HeLa cells. The microscopy data suggested that the migration of the POSS_F molecules to the cell cytoplasm was time-dependent and driven by the high affinity of the POSS to the membrane double layer of HeLa cells. In addition, inhibition experiments of different endocytic pathways indicated that POSS_F molecules are endocytosed via the macropinocytosis route.Inspired by Rotello’s reports on the use of POSS for bioimaging applications, Marchese and collaborators prepared a novel, highly luminescent bifunctional POSS containing its their structure a fluorescein derivative (FITC) as a fluorophore (POSS_F) and a carboxylic functionality ready to anchor several organic/inorganic molecules for biomedical applications . The strMiksa and coworkers have developed biocompatible POSS-based phenosafranin dye (PSF) nanohybrids using a coupling chemistry for drug delivery and diagnostics . PSF is Liu et al. reported on the development of a three-dimensional, water-soluble, hybrid nanodot based on POSS and a conjugated oligoelectrolyte (COE) for two-photon excited fluorescence (TPEF)  62]. In. In62]. Wang and his group have explored the use of octaammonium-POSS (OA-POSS) to functionalize the surface of carbon dots (CDs). Bare CDs without surface functionalization or passivation generally exhibit very weak emissions in aqueous medium and other solvents. The passivation is carried out through the electrostatic interaction between the ammonium groups of OA-POSS and the carboxylate groups on the surface of CDs . As a reRare earth-doped upconversion nanoparticles (UCNPs) are considered a new promising generation of imaging agents for bioimaging ,107. UCNIn this review, we highlighted recent research progress on the utilization of polyhedral oligomeric silsesquioxane for biomedical application with an emphasis on drug delivery, photodynamic therapy and bioimaging. In addition, the different strategies for the functionalization of POSS; in particular, the use of click chemistry was summarized. The use of POSS as delivery platform is a burgeoning area of research, which was demonstrated in this review with illustrative examples using anticancer drugs, genes, photosensitizers and contrast imaging agents. Low toxicity, biocompatibility, stability and well-established functionalization techniques are critical features that make POSS attractive in this field. In addition, there are key benefits for the use of POSS in photodynamic therapy: the rigid cage structure acts as a spacer molecule, preventing the aggregation-induced quenching effect which is typically associated with the poor performance of photosensitizers, and POSS can be used as a template to control photosensitizer placement after the nanoparticles have been produced. Finally, the use of POSS as a coating agent for nanoprobes not only renders functionalization capabilities to the material, but can also be used as an effective passivating agent reducing surface trap states that can quench photoluminescence.While the current results are encouraging and show great potential for future applications of POSS in biomedicine, key breakthroughs are still needed to move this platform forward to clinical applications. The regioselective functionalization of POSS is still a challenge. Novel chemical approaches with controlled and efficient synthesis of POSS that render precise placement of therapeutic/contrast agent and/or functional groups in the molecule are worth developing. Contrary to other silica-based materials such as mesoporous silica or polysilsesquioxane nanoparticles that tend to accumulate in organs like the liver or spleen, ,87,92, iWe envision that basic understanding and applications of POSS in the area of biomedicine will continue to grow offering future breakthroughs on the synthesis of new POSS platforms, and the clinical implementation of this technology will be developed.


In response to the current common disease of concrete leveling overlays of bridge pavement in China, the feasibility of using an economic SSPP-ECC with local waste superfine sand as an alternative material for a leveling overlay was proposed in this study. To evaluate the interface bonding property in the girder between the SSPP-ECC and concrete, a slant shear test and split tensile test were designed to study the interfacial shear and tensile properties of the ordinary concrete/ordinary concrete (OC/OC) and ordinary concrete/SSPP-ECC (OC/ECC), where the results showed that SSPP-ECC could significantly improve the interface shear stress and split tensile strength compared to ordinary concrete. Furthermore, the damage status of OC/ECC no longer involved fracturing along the interface; instead, each of the two substrates was partially destroyed, which revealed that OC/ECC had a high bonding effect. Moreover, a restrained shrinkage test was carried out to evaluate the shrinkage property of SSPP-ECC, where the result showed that the shrinkage strain of SSPP-ECC was slightly lower than concrete, where the average cracking time for SSPP-ECC was far longer than for ordinary concrete under the same ambient drying conditions; furthermore, the stress rate for SSPP-ECC revealed that it was a low-cracking-risk material. Meanwhile, the crack width of SSPP-ECC was only 0.1 mm after 35 d, which showed that SSPP-ECC had a more substantial crack width control capacity relative to concrete. The test results initially verified the feasibility and great potential of economic SSPP-ECC applied in a bridge pavement leveling overlay. Bridge pavement is a typical multi-layer structure with different materials. The conventional structure of current bridge pavement consists of a composite asphalt surface layer, a binding layer, and a waterproof leveling layer ,2,3. DueThe leveling overlay in bridge pavement is similar to the concrete overlay used for bridge deck repair and reinforcement methods for the laying position and thickness . As the Since ECC was invented in the 1990s, scholars have carried out several studies on its properties, and it is still one of the popular cementitious materials ,22,23. EIn summary, taking SSPP-ECC as a material in bridge pavement may provide better effects. A series of studies were carried out to explore the feasibility of SSPP-ECC used in leveling overlays. First, the basic mechanical properties of SSPP-ECC were tested to evaluate the feasibility of applying it to the leveling overlay. Second, the interfacial bonding of the shear stress and the split tensile strength were analyzed to evaluate the bonding properties of SSPP-ECC with ordinary concrete. Finally, considering the fact that the casting area of the leveling overlay covered the whole bridge deck and the proportion of cementitious binders of ECC was high, a restrained shrinkage test was carried out to study the shrinkage properties of SSPP-ECC during the hardening stage. Thus, the tests in this study were undertaken to explore the feasibility of SSPP-ECC as a new material for leveling overlays and to verify its critical application properties, which have profound research significance. It is hoped to minimize the occurrence of diseases associated with leveling overlays and to contribute to the application of functional cementitious composite material in bridge pavements.The constituent materials of SSPP-ECC were all fine sized, i.e., cementitious binders, PP fiber 30 μm in diameter and 12 mm in length, local superfine river sand with a maximum particle size of 0.6 mm, and water, which obviously has better adaptability to some thin pouring condition in the leveling overlay than ordinary concrete with coarse aggregates. The cementitious binders included the ordinary Portland cement 52.5 R and F.II grade fly ash produced by Jilin Yatai Group , whose components and properties are shown in The mix proportion of SSPP-ECC was determined based on the existing research in the group and ordinary concrete was formulated with C50 as the target ,44. The The preparation processes for the SSPP-ECC were as follows: first, cement, fly ash, and some of the water (to give a water–cement ratio of 0.26) were added while mixing slowly for 1 min; second, PP fiber and HRWR were added with slow mixing for 1 min and then mixed quickly for 2 min. Finally, superfine river sand and the remaining water were added with slow mixing for 1 min and then mixed quickly for 3 min to complete the mixing, as shown in To compare with the concrete that is currently widely used in leveling overlays, the basic mechanical properties of SSPP-ECC were tested first. According to the Chinese specification JTG 3420—2020 , the comnτ—shear stress (MPa);P—maximum applied compressive force (kN);A—cross-section angle (mm2);α—interface angle (30°).For the bonding interface between the leveling overlay and the girder, the main damage is caused by shear or tensile stresses. The slant shear test was carried out to evaluate the interfacial shear resistance property between SSPP-ECC and ordinary concrete. The specific specimen size is shown in sf—split tensile strength (MPa);P—maximum applied load (kN);A—area of the bonded load (mm2).The split tensile test was carried out to evaluate the interfacial tensile property between SSPP-ECC and ordinary concrete. The specific specimen size is shown in Under conventional service conditions, cracks in bridge decks are caused by temperature changes, shrinkage in concrete, etc. Among these factors, shrinkage in concrete had the most significant impact on the development of cracks ,47. Conscrt and the average stress rate S were used to evaluate the cracking resistance. The cracking age was easily obtained from a moment of an abrupt change in the strain–time curve. The crack number and crack distribution were recorded, and the crack observation instrument was used to measure the crack width, as shown in netε was calculated as the difference between the strain in the steel ring at each recorded time and the initial time. The elapsed time t was calculated as the difference between each recorded time and the initial time. Plotting the net strain against the square root of the elapsed time for each strain gauge on the test specimen and using linear regression analysis to fit a straight line through the data. The strain rate factor was the slope of the line, according to Equation (3):netε—net strain (m/m);1/2);α—strain rate factor for each strain gauge on the test specimen ((m/m)/dt—elapsed time (d);k—regression constant.According to ASTM C 1581-04, the net cracking age α from each strain gauge, these values were transformed into the average rate factor |avgα| of two strain gauges and the stress rate q was calculated according to Equation (4):q—stress rate in each test specimen (MPa/d);G = 10.47 × 106 psi (72.2 GPa);avg|—absolute value of the average strain rate factor for each test specimen ((m/m)/d1/2);|αrt—elapsed time at cracking or elapsed time when the test was terminated (d).After obtaining the rate factor S, the shrinkage cracking risks of the SSPP-ECC and ordinary concrete were evaluated.Finally, the average stress rate for the test specimens was calculated with an accuracy of 0.01 MPa/d. According to the value of The results of the comparison of the basic mechanical properties between ordinary concrete and SSPP-ECC are shown in The slant shear test reflects the interfacial shear resistance; the resulting load–deformation curves are shown in During the loading process of the slant shear test, the absorption energy was determined using the area under the load–deformation curve shown in The split test reflects the interfacial tensile property. The load–deformation curves are shown in During the loading process of the split tensile test, the absorption energy was determined by the area under the load–deformation curves shown in −6 growth strain before both entered a low-rate growth phase. It can be seen that the shrinkage strain growth rates of the two materials had apparent differences, where the concrete’s properties led to the strain growing faster. The strain–time curve changed abruptly at about 7 d, where noticeable cracks could be observed on the surface of the specimen, as shown in −6, while the average maximum strain of SSPP-ECC was 86.85 × 10−6, indicating that the strain of SSPP-ECC was slightly lower than that of concrete. Although SSPP-ECC used fewer aggregates, the high fly ash content significantly slowed the volumetric shrinkage in the hydration stage. Meanwhile, the bond strength between the fibers and matrix gradually grew. The resistance to shrinkage cracking was steadily enhanced; therefore, the shrinkage cracking time of SSPP-ECC was much longer than that of concrete. It was concluded that SSPP-ECC had better volume stability.The statistical results of the maximum strain and the cracking time in the steel ring by the two materials are shown in To compare the shrinkage crack development conditions of the two materials, the crack width of each group was measured using a crack observation instrument every 7 d. The average crack width results are shown in S was calculated for each group according to Equations (3) to (4). The cracking risk can be evaluated according to ASTM C 1581-04. The results showed that the average cracking time for concrete is 6.5 d, while the average stress rate S was 0.595, which meant that concrete was a high-cracking-risk material. In contrast, the average cracking time for SSPP-ECC was 27 d, which was not above 28 d to qualify as low cracking risk. While its average stress rate S was only 0.073, it was sufficient to meet the requirements of low cracking risk for stress rate such that SSPP-ECC can be considered a low cracking risk material.As shown in q—interface stress;1E—Young’s modulus of ordinary concrete/SSPP-ECC;2E—Young’s modulus of the steel ring;1μ—Poisson’s ratio of ordinary concrete/SSPP-ECC;2μ—steel ring Poisson’s ratio;θσ—outer ring tensile stress;θε—inner ring shrinkage strain;shε—outer ring shrinkage strain.The strain value measured on the surface of the inner ring in the restrain shrinkage test is theoretically related to the actual shrinkage deformation and tensile stress . ReferriThe average tensile stress on the outer surface of the specimens can be obtained from the association of Equations (5) and (6), and the shrinkage deformation on the outer surface of the specimens can be obtained from the association of Equations (5) and (7). Since the concrete specimens already reached the maximum shrinkage strain and cracked after 7 d, while the SSPP-ECC specimens had the maximum shrinkage strain after approximately 27 d, the average tensile stress and shrinkage deformation on the outer surface of the concrete at 7 d and the SSPP-ECC at 7 d and 27 d are shown in −6 at 7 d, while the SSPP-ECC was slightly lower than concrete with 618 × 10−6. After that, the concrete specimens showed larger shrinkage cracks, while SSPP-ECC specimens continued to resist shrinkage and the shrinkage strain only increased by 14% after 27 d. More importantly, the maximum tensile stress at the outer surface of the concrete specimens at 7 d was 11.930 MPa, while in SSPP-ECC, the tensile stress was 9.061 MPa, and even the maximum tensile stress of SSPP-ECC specimens after the shrinkage time reached 27 d was lower than the maximum tensile stress in concrete at 7 d, indicating that the SSPP-ECC had stronger resistance to shrinkage and cracking. The tensile stress of SSPP-ECC shrinkage was more minor; therefore, the problem of excessive local stress caused by shrinkage is less likely to occur when casting SSPP-ECC in leveling overlay.As shown in To evaluate the feasibility of economic SSPP-ECC as an alternative material for bridge pavement leveling overlay, the authors conducted an experimental analysis of its interfacial bonding effect and resistance to shrinkage cracking. The main conclusions were as follows:The slant shear test results showed that interfacial scratch treatment provided a better enhancement effect on split tensile strength and using SSPP-ECC instead of ordinary concrete in shear stress provided more significant enhancement than a scratch treatment. Moreover, the damage status of OC/ECC-Y was no longer completely damaged along the bonding interface as in other specimens, which showed that OC/ECC-Y had an excellent shear resistance property.The split tensile test results showed that the interfacial scratch treatment provided an enhancement effect regarding split tensile strength, while the lifting effect was low relative to the shear stress; using SSPP-ECC instead of ordinary concrete provided a more significant enhancement in split tensile strength than a scratch treatment. The damage status of OC/ECC-Y was no longer showed complete damage along the bonding interface as in other specimens, which showed that OC/ECC-Y had excellent tensile resistance.The restrained shrinkage test showed that the average cracking age of SSPP-ECC was far longer than ordinary concrete, which showed that SSPP-ECC could significantly delay the cracking time. The shrinkage strain of SSPP-ECC was slightly lower than concrete, and its average stress rate confirmed SSPP-ECC as a low-cracking-risk material. Moreover, the crack width of SSPP-ECC was far lower than ordinary concrete, which confirmed SSPP-ECC had a powerful crack width control capacity.In summary, SSPP-ECC had a proficient bonding effect with ordinary concrete, which had stronger resistance in shear and tension. SSPP-ECC had better volume stability and substantial shrinkage cracking resistance. Thus, SSPP-ECC can be applied to the construction of a large area and low thickness of the leveling overlay in bridge pavement.In this study, the feasibility of economical SSPP-ECC as a leveling overlay material for bridge pavement was investigated through a laboratory test, which initially revealed that SSPP-ECC had a stronger interfacial property and shrinkage cracking resistance compared to ordinary concrete. More work will be completed in the future, such as conducting a detailed numerical simulation analysis, fatigue test analysis under impact loading, and more practical engineering studies. These are the main contents that the author will carry out in subsequent experimental designs.


To compare antibiotic sales in eight high-income countries using the 2019 World Health Organization (WHO) Access, Watch and Reserve (AWaRe) classification and the target of 60% consumption of Access category antibiotics.We analysed data from a commercial database of sales of systemic antibiotics in France, Germany, Italy, Japan, Spain, Switzerland, United Kingdom of Great Britain and Northern Ireland, and United States of America over the years 2013–2018. We classified antibiotics according to the 2019 AWaRe categories: Access, Watch, Reserve and Not Recommended. We measured antibiotic sales per capita in standard units (SU) per capita and calculated Access group sales as a percentage of total antibiotic sales.In 2018, per capita antibiotic sales ranged from 7.4 SU (Switzerland) to 20.0 SU (France); median sales of Access group antibiotics were 10.9 SU per capita (range: 3.5–15.0). Per capita sales declined moderately over 2013–2018. The median percentage of Access group antibiotics was 68% (range: 22–77 %); the Access group proportion increased in most countries between 2013 and 2018. Five countries exceeded the 60% target; two countries narrowly missed it . Sales of Access antibiotics in Japan were low (22%), driven by relatively high sales of oral cephalosporins and macrolides.We have identified changes to prescribing that could allow countries to achieve the WHO target. The 60% Access group target provides a framework to inform national antibiotic policies and could be complemented by absolute measures and more ambitious values in specific settings. We analysed wholesale antibiotic sales data for 2013–2018 from eight countries to determine patterns of sales with reference to the 2019 WHO AWaRe classification.,,,,We used the IQVIA multinational integrated data analysis system database  to identify antibiotic sales. This commercial database tracks pharmaceutical sales by using national sales audits of manufacturers and wholesalers, through retail and non-retail channels.,,To extract data on systemic antibiotic formulations from the sales database, we developed a comprehensive list of antibiotics used in human medicine. We used three sources: (i) WHO list of critically important antimicrobials for human medicine, 2018 edition; (ii) WHO anatomical therapeutic chemical (ATC) code J01 ; and (iii) WHO AWaRe classification, as presented in the 2019 WHO essential medicines list and in the WHO AWaRe classification database.,We estimated sales volumes in standard units (SU). SU refers to the number of standard dose units sold, where a dose is defined by IQVIA as one tablet or capsule for solid forms, one ampoule or vial for injectable forms, and 5 mL for syrup forms. We aggregated data at the year level by country and product. We defined each antibiotic product as Access, Watch, Reserve or Not Recommended, according to the 2019 AWaRe categorization.We used several metrics to explore sales patterns . First, We compiled additional data to analyse between-country differences in antibiotic sales. Country-level sociodemographic indicators were obtained from the World Bank.As of May 2020, all countries had implemented an antimicrobial national action plan and three countries had adopted AWaRe for antibiotic use surveillance  of total sales in Japan to 76.8% in the United Kingdom (1000/1302 million SU) and 77.2% in the USA ; amoxicillin/clavulanic acid sales were ≥ 3% in all countries except Japan  as shown in the data repository.For external validity, we compared comparable country–year results against European Centre for Disease Prevention and Control network data for France, Italy, Spain and the United Kingdom. These results showed a strong correlation and support the validity of our results (see the data repository).It is encouraging that in 2018 several of the countries studied achieved the WHO 60% Access group target, with a median percentage sales of Access group antibiotics across the eight countries of 68%. Of the three countries not meeting the target, Italy and Germany narrowly missed it, while Japan’s proportion was notably lower. Most countries made progress in optimizing antibiotic sales between 2013 and 2018, both in terms of an increase in the relative sales of Access group products and a decrease in per person sales of antibiotics.,,,The AWaRe classification and associated target provides a framework for simplified and standardized antibiotic surveillance and has the support of the G20 group of governments and central bank governors.,,,In 2018, WHO released the first global report applying the 2017 AWaRe classification to evaluate 2015 levels of antibiotic use.,,Japan stands out as having a differing pattern of antibiotic consumption. We found lower relative sales of Access group broad-spectrum penicillins and higher sales of Watch category antibiotics, predominantly driven by high relative sales of third-generation cephalosporins and, to a lesser extent, macrolides and fluoroquinolones. These findings corroborate other studies of antimicrobial sales in Japan.The 60% Access group target provides a simple metric to monitor Access and promote responsible antibiotic use, but an emphasis on relative consumption alone could have unintended consequences on absolute consumption. In Germany, the Access group target was narrowly missed. The target could be achieved by a switch from second-generation Watch group cephalosporins to the Access group first-generation cephalosporins, but likewise it could be met through unnecessarily increasing sales of Access group products. Japan, despite a low Access group index, had relatively low total antibiotic sales and relatively low reported rates of antibiotic resistance. The European Centre for Disease Prevention and Control list of indicators for monitoring antibiotic consumption utilizes an absolute measure as the primary indicator of consumption to consider the amount of antimicrobials used.,,,Our study had some limitations. We estimated sales using SU, a standardized measure within the data source representing a single dose unit of sales. This method differs from the WHO consumption surveillance, which uses the ATC/defined daily dose method of calculation.The use of sales data has certain limitations. Foremost, we did not study individual-level consumption. We could not determine patient characteristics and indications for treatment: factors critical to determining the appropriateness of prescribing and antibiotic use. Second, sales data may not be representative of the entire market – particularly for those countries where the data covers less than 100% of the pharmaceutical market – and the IQVIA algorithm to produce nationally representative estimates is not publicly available. Third, it may not be possible to disaggregate data by sector, facility and subnational geographies. Finally, the use of aggregate sales data provides a simple and standardized proxy for antibiotic consumption but should be complemented by analysis of data sources that enable conclusions to be drawn about the appropriateness of antibiotic use at the patient level.Finally, while including a smaller subset of countries has allowed for a more in-depth analysis of antibiotic sales practices in this selection of highly developed countries, the findings need to be complemented by global data and more heterogeneous settings. Global increases in antibiotic consumption have been shown to be driven by rapid increases in consumption of Watch group antibiotics, particularly in low- and middle-income countries, highlighting the need for broader analyses to support stewardship efforts.Monitoring antibiotic consumption is an essential policy action highlighting potential areas where changes are needed to reduce the risk of antimicrobial resistance. As countries adopt the WHO AWaRe framework, there is a need to assess changes in antibiotic sales and use over time, and whether the WHO Access target is sufficient to preserve antibiotic efficacy across a range of infections or should be expanded. All countries should consider adapting the AWaRe classification and target to individual settings, with country-specific targets unambiguously reported in antimicrobial national action plans. Additional metrics, such as those focusing on absolute consumption, and more ambitious targets are needed to better adapt appropriateness of antibiotic use, particularly in mature health-care systems.


E. coli BL21(DE3) exclusively based on bioengineering methods.In many industrial sectors continuous processing is already the golden standard to maximize productivity. However, when working with living cells, subpopulation formation causes instabilities in long-term cultivations. In cascaded continuous cultivation, biomass formation and recombinant protein expression can be spatially separated. This cultivation mode was found to facilitate stable protein expression using microbial hosts, however mechanistic knowledge of this cultivation strategy is scarce. In this contribution we present a method workflow to reduce workload and accelerate the establishment of stable continuous processes with  Escherichia coli is frequently employed in research and industry for the production of recombinant proteins 3. The advantages of a large toolbox for genetic modifications as well as fast doubling times outweigh the negative traits, such as the lack of posttranslational modifications 6. Lactose and the synthetic structural analogue IPTG  are common transcription inducers for T7-based E. coli expression hosts, like BL21(DE3) 9. Whereas IPTG is known to cause cellular stress ensuing toxic effects at concentrations higher than 1 mM 9, no such effects have been reported for lactose 12. High concentrations of recombinant product additionally have been reported of causing host cell toxicity 13, potentially favoring the use of lactose over IPTG due to slower heterologous protein expression. Furthermore, induction by lactose increases the production of soluble product 16. Recently, we reported that co-utilization of glycerol and lactose promotes recombinant protein expression and increases viability compared to a mixed-feed with glucose 18. Unlike glucose, glycerol is integrated into glycolysis and gluconeogenesis 18. Results of other studies also indicate altered TCA-activity on glycerol compared to glucose metabolism potentially favoring recombinant protein production 21.E. coli bioprocesses it is known that batch and fed-batch cultivations result in unwanted variable, time-dependent productivity 22, however these processing modes are still state-of-the-art for industrial applications. Time-independent processing would reduce batch to batch variations and consequently allow stable productivity and robust downstream processing 24. Furthermore, a change from fed-batch to continuous manufacturing would allow a reduction in scale and operating costs and thus should be pursued as the most efficient cultivation technique 27. However, solutions for continuous cultivation with E. coli have not been realized yet, since subpopulations evolve over elongated cultivation times 30, which are yet to be investigated 31.For 34. Even though a spatial separation of biomass formation and recombinant protein production seems promising, the effects of process parameters on productivity in such a cascaded continuous cultivation are barely known 35. Further, long-term effects beyond 100 h of induction and thus the potential for continuous manufacturing with E. coli have not been investigated yet 36 because the development of continuous processes is highly time- and resource-dependent.Previous studies highlighted a cascaded continuous operating system using two bioreactors, to reduce subpopulation formation compared to common continuous cultivations  E. coli host over extended cultivation times of more than 220 h using cell specific productivity as a key performance indicator to determine cell equilibrium state. Glycerol-lactose systems were utilized solely for continuous process development, due to their beneficial results over glucose-fed and IPTG-induced systems in diverse pre-studies37. After intensive screening and variation of selected process parameters, a workflow on how to set-up continuous cultivations with E. coli BL21(DE3) was developed, tested and verified. Consequently, we present a first workflow to set up and accelerate stable continuous process development with E. coli BL21(DE3).In this study, we investigated the cascaded continuous cultivation with a T7-based E. coli BL21(DE3) was set up as shown in Fig. p [mg/g/h]) and space time yield (STY [mg/L/h]). The STY was chosen as indicator to determine the volumetric product throughput, which is frequently used to evaluate continuous processes. The process parameters dilution rate and glycerol-lactose mixed feed ratio were varied according to a multivariate design  strain producing a N-Pro fusion protein. Essential physiological parameters, which are required for process development, have been determined in previous studies and are summarized in Table To screen the expression of recombinant proteins in a cascaded continuous operating system, we used a recombinant 34. As a high number of variables in a multivariate design space would result in a remarkably high experimental load, we started with univariate experiments varying the specific glycerol uptake rate qs,gly during induction, by keeping the total dilution rate constant . The ot18. All performed cultivations followed the same trend: STY and qp reached a maximum after the start of induction and then decreased after 20 generations , however STY and qots Fig. . As proc.72 Fig. .Figure 3p. We included the star point experiments in the multivariate design and modeled the response in terms of STY and qp , which led to stable long-term protein expression. In a next step, we summarized our findings in a workflow protocol which comprises two phases.As µFor the strain producing the N-pro fusion protein essential physiological data was already known from previous studies Table . To detes,lac) are determined. Maximum growth rate is easily available upon a batch fermentation with excess carbon source. After the batch, lactose is pulsed in the reactor and uptake of lactose is monitored in a timely resolved way, e.g. by sampling every 30 min  the maximum growth rate and specific inducer uptake rates  must be determined. This can be done in a series of static experiments: A fixed qs,gly is applied, inducer is pulsed in excess and lactose decrease in the broth is measured 42. However, using this approach a high number of cultivations is necessary. We recently showed that this relation can be obtained more elegantly in only three experiments using dynamic feeding during induction 42. Therefore, in step (II) a fed-batch is performed at qs,gly of 0.1 g/g/h to determine the maximum qs,lac   were found to result in high recombinant protein production in fed-batches 43. Hence, we recommend to vary the feed ratio for screening near to the maximum inducer uptake rate, to prevent inducer deficiencies and also investigate moderate to high sugar uptake rates .As intensive screening of cascaded continuous cultivation has been conducted for the N-pro protein, we hypothesize that determined dilution rate optima for the N-pro protein can be transferred to the expression of other proteins using glycerol as C-source, reducing the experimental workload. Based on the previous findings, we recommend to alter the dilution rates in a narrow range of 0.16 to 0.22/h. In addition moderate to high uptake rates (qE. coli BL21(DE3) using glycerol as carbon source and lactose as inducer, can be summarized as: In phase I physiological parameters are determined , chosen process parameters had a comparable impact on specific productivity Fig. b. Model The proposed workflow Figs. , 5 thus We want to stress that the described process development for cascaded bio-manufacturing could be solely done with process engineering methods. This technique is thus ideal for being transferred to process development laboratories in industry. To strengthen this hypothesis STY from both processes were compared to state-of the art fed-batch cultivations which is the industrial golden standard. Total amount of produced protein was calculated for cascaded continuous process and fed-batch  was not within the scope of this study. In fact, for one of the chosen products such analytics would not have been technically feasible. In this study, we propose a workflow on how to set up continuous cultivations, keeping cells in an equilibrium state for induction times longer than 220 h using glycerol and lactose. Choosing recombinant protein titer (shown as qp and STY) to determine cell equilibrium state in continuous cultivation, negligible long-time effects were observed at optimized process conditions. Results indicate low dilution rates (D = 0.1/h) to be unsuitable for achieving long-term stable productivity, independent from the applied uptake rates 34. We believe that high residence times (resulting from low dilution rates) enhance subpopulation diversification, as cells have more time to adapt to environmental conditions and to evolve a non-producing sub-population 46. High dilution rates (implementing low residence times) were found to result in stable productivity. The principle facilitates continuous bio-manufacturing, as higher volumetric rates would lead to higher amount of bleed and consequently higher STY. Even though cultivations at higher dilution rates, such as D = 0.24/h and D = 0.3/h were found to result in stable productivity . We suggest to screen initial physiological parameters in a batch and two fed-batch runs (phase I) at first. Based on the initial screening experiments and the determination of physiological parameters (output of phase I) a reduced experimental design can be used to optimize process conditions in cascaded continuous cultivation (phase II). Testing the anticipated workflow showed that identical process parameters led to best results, independent from the recombinant protein. Stable specific productivity at highest space–time yield for the expression of both target proteins was found at a dilution rate of 0.22/h conducted at a feed ratio of 2 . Even tough ideal cultivations were well comparable for both proteins exploitation of different carbon sources and inducers could lead to different effects .As the ideal operating space for recombinant protein expression in cascaded continuous processing was intensively screened, obtained results were taken to propose a generic workflow for the establishment of cascaded continuous cultivation for 10, we were able to establish stable continuous cultivations for a new target product within less than a month. Results presented in this study also show that cascaded continuous cultivation is superior to fed-batch cultivation for E. coli BL21(DE3) and hence is a suitable tool for bio-manufacturing. Consequently, the demonstrated experimental workflow could be used to set up and accelerate the implementation of a stable continuous upstream process for E. coli.Still, for new BL21(DE3) strains and products, we propose an experimental workflow with two phases using solely bioengineering methods. As parallelization of continuous processes can be used to speed up process development E. coli strain BL21(DE3). The protein used for initial studies is an industrial protein (28.8 kDa), being linked to an N-pro fusion tag and integrated into a pET-30a plasmid system 38. The protein used for verification and transfer studies was mCherry, being integrated also in a pET30a plasmid system. Expression for mCherry was dominantly soluble in the cytoplasm, with small fractions being expressed as an inclusion body.All performed cultivations were carried out using the 47.For all cultivations, a defined minimal medium referred to DeLisa et al. was used  contained 25 g/L of glycerol, while the mixed feed was altered according to the DoE approach . The batch medium was inoculated the next day with the preculture using 20% of the reactor volume.34. In each run, one reactor was used for biomass formation and the derived biomass stream was transferred to the second reactor, controlled via pumps monitored by the process system PIMS Lucullus . Biomass formation (reactor 1) was performed in a Labfors 3 bioreactor . The reactor was coupled to a continuously operated stirred-tank reactor—, used for recombinant protein expression. Reactor 1 was aerated with a mixture of pressurized air and pure oxygen at 3 vvm and constantly stirred at 1000 rpm. Minifors reactors were aerated with 2 vvm and stirred at 1400 rpm. The off-gas concentrations of CO2 and O2 were monitored via BlueSens Gas sensors . Throughout all cultivations the dissolved oxygen (dO2) was kept above 40% by adjusting the ratio of pure oxygen and pressurized air. The dissolved oxygen was monitored with a fluorescence dissolved oxygen electrode Visiferm DO425 . As throughout the continuous phase the volume in the reactors was controlled with a dip pipe adjusted to the liquid surface, no stirrer cascade was implemented. pH was maintained at a constant value of 6.7 through all process phases and controlled by addition of NH4OH (12.5%). The pH was monitored with an EasyFerm electrode . In all three reactors, a batch was conducted with carbon concentrations of 20.4 g/L, yielding in 9–10 g dry cell weight of biomass per litre of cultivation broth. The end of the batch phase was determined by a drop in the CO2 signal.The used set up for the cascaded continuous cultivation is sketched in Fig. 22, whereas 30 °C where applied throughout induction phase for mCherry, due to previous results obtained for soluble protein expression 43.Afterwards, the non-induced chemostat system was started by supplying the biomass reactor with a dilution rate of 0.14/h feed 1  was adjusted to the desired dilution rate according to the design of experiment.The so-called continuous adjustment phase and lasted over-night to achieve a steady state in all reactors, monitored by an equilibrium state of reactor 1 of derived pOs,gly of about 0.15 g/g/h a static experiment was performed using a classic fed-forward approach and lactose pulses. Higher qs,gly were screened using a linear ramp from about qs,gly 0.25 to 0.55 g/g/h of the glycerol feed in combination with lactose pulses. While for BL21, uptake rates are similar 42, results might be different for other E. coli strains using lactose as inducing agent.Experiments to determine uptake rates in mCherry, were performed in a Sartorius Biostat Cplus bioreactor  with 10 L working volume. Monitoring and control were performed via Lucullus . The maximum glycerol uptake rate was determined in regularly sampling during the batch. Afterwards a lactose was added via pulse and uptake of solely lactose was evaluated. At a qs,C. Feed addition was started after the end of the batch, indicated by a drop of the CO2 signal. Feeding was conducted at qs = 0.2 g/g/h and lasted over-night to generate 25–30 g/L biomass. Prior to induction temperature was adjusted to 31.5 °C for Npro and to 30 °C for mCherry. The exponential feeding was set to a qs = 0.3 g/g/h for both proteins. Induction was performed via a 0.5 mM IPTG pulse to the fermentation broth and induction lasted for 10 h.Pre-culture and process analytics were performed equivalent to the cascaded continuous cultivations. Fed-batches were conducted with an exponential feeding approach at a constant q2), the model predictability (Q2), the model validity and the model reproducibility, adjusted for degrees of freedom. Analysis of the process dataset was done by a multivariate data assessment . Time independent rates (space–time yield STY (mg/L/h) and specific productivity qp (mg/g/h)) were chosen as responses to be evaluated for this study.Multivariate data assessment of the performed experimental design was performed. Results were investigated for the statistical relevance of the model using the measure of fit . Time independent rates (specific productivity qp (mg/g/h) and space–time yield STY (g/L/h) were chosen as model responses to be evaluated for this study. Rates were calculated as stated beneath:Process analytics were performed as described in previous studies p: specific productivity [mg/g/h], qSpace–time-yield used for comparison, was calculated by the following Eqs.  and 3)::3):2\docmentclass2pt{minimmentclass2pt{minimSupplementary Information.