Serum Proteomic Profiles of Patients with Chronic Recurrent Multifocal Osteomyelitis.

OBJECTIVES: Chronic recurrent multifocal osteomyelitis (CRMO) is an autoinflammatory disease characterized by sterile bone inflammation; however, its pathophysiology is poorly understood. Thus, this study aimed to characterize the serum proteomic profiles of patients with CRMO to better understand the molecular mechanisms underpinning CRMO pathogenesis. METHODS: Proteomic profiling of the sera collected from 11 patients with CRMO (five patients were in active phase, six were in inactive phase) was conducted using liquid chromatography-mass spectrometry. Sera from four children without inflammatory diseases were used as controls. Pathway analysis was performed to identify the upregulated and downregulated proteins in patients with active CRMO. RESULTS: Compared with the control group, 19 and 41 proteins were upregulated and downregulated, respectively, in patients with active CRMO. Pathway and process enrichment analyses revealed that axon guidance was the most enriched category of upregulated proteins in patients with active CRMO, followed by neutrophil degranulation and mitogen-activated protein kinase cascade regulation. In comparison to patients with inactive CRMO, 36 proteins, including 11 keratin proteins, were upregulated and highly enriched in the intermediate filament organization category. Rho GTPase pathway-related proteins were downregulated in ibuprofen-treated patients. CONCLUSION: Proteomic analysis identified upregulated proteins in the sera of patients with acute CRMO. These proteins can be used as biomarkers for disease diagnosis and activity. Furthermore, we anticipate that this study will contribute to a deeper understanding of the pathophysiology of CRMO, which, in turn, will contribute to the discovery of potential novel therapeutic targets.

Association between adeno-associated virus 2 and severe acute hepatitis of unknown etiology in Japanese children.

INTRODUCTION: Outbreaks of acute hepatitis of unknown etiology (AHUE) in children were reported in Western countries in 2022. Previous studies found that adeno-associated virus 2 (AAV2) and its helper viruses, such as human adenovirus (HAdV) and human herpesvirus-6 (HHV-6), are frequently detected in patients with AHUE. However, the existence of hepatitis associated with AAV2 prior to AHUE outbreaks in 2022 had not yet been investigated. We aimed to investigate the association between AAV2 and pediatric acute hepatitis in Japanese children, as well as the incidence of AAV2-related hepatitis prior to 2022. METHODS: Preserved blood samples obtained from 49 pediatric patients with acute hepatitis between 2017 and 2023 were retrospectively analyzed. Blood samples from 50 children with acute illnesses and 50 children with chronic conditions were used as controls. Viral DNA loads were quantitated using real-time PCR. RESULTS: AAV2 DNA was detected in 12 % (6/49) of acute hepatitis cases but in only one acute illness and none of the chronic-condition control cases. The concentration of AAV2 DNA in the six acute hepatitis cases was higher than that in the acute-illness control case. Co-infection with one or more helper viruses, including HAdV, HHV-6, cytomegalovirus, and Epstein-Barr virus, was observed in five AAV2-positive cases. CONCLUSIONS: Our results indicated the sporadic occurrence of pediatric severe hepatitis associated with AAV2 infection in Japan prior to the AHUE outbreaks in 2022. Our findings suggest that co-infection with AAV2 and helper viruses plays a role in developing severe hepatitis.

Comparison of Whole Blood and Plasma for Monitoring Cytomegalovirus and Epstein-Barr Virus.

Monitoring Epstein-Barr virus (EBV) and cytomegalovirus (CMV) infection after transplantation is recommended to enable preemptive therapy. However, the most suitable sample type remains unclear. Patients who underwent hematopoietic stem cell or liver transplantation were included in this study. Viral loads in sequential whole-blood and plasma samples were retrospectively analyzed. EBV DNA was detected more frequently in whole blood (55%) than in plasma (18%). The detection rate of CMV DNA was similar between the two sample types. The correlation of viral loads between the two sample types were 0.515 and 0.688 for EBV and CMV, respectively. Among paired samples in which EBV DNA was detected in whole blood, the plasma EBV detection rate was significantly higher in patients who underwent hematopoietic stem cell transplantation than in those who underwent liver transplantation. The viral DNA load in whole blood and plasma showed similar trends. The EBV detection rate was higher in whole blood, and a high correlation was observed between CMV DNA loads and whole blood and plasma. These results indicate that whole blood is more sensitive for monitoring both EBV and CMV, whereas plasma is a potential alternative sample for monitoring CMV.

Purification and partial characterization of the extradiol dioxygenase, 2'-carboxy-2,3-dihydroxybiphenyl 1,2-dioxygenase, in the fluorene degradation pathway from Rhodococcus sp. strain DFA3.

Type II extradiol dioxygenase, 2'-carboxy-2,3-dihydroxybiphenyl 1,2-dioxygenase (FlnD1D2) involved in the fluorene degradation pathway of Rhodococcus sp. DFA3 was purified to homogeneity from a heterologously expressing Escherichia coli. Gel filtration chromatography and SDS-PAGE suggested that FlnD1D2 is an α4ß4 heterooctamer and that the molecular masses of these subunits are 30 and 9.9 kDa, respectively. The optimum pH and temperature for enzyme activity were 8.0 and 30 °C, respectively. Assessment of metal ion effects suggested that exogenously supplied Fe(2+) increases enzyme activity 3.2-fold. FlnD1D2 catalyzed meta-cleavage of 2'-carboxy-2,3-dihydroxybiphenyl homologous compounds, but not single-ring catecholic compounds. The Km and kcat/Km values of FlnD1D2 for 2,3-dihidroxybiphenyl were 97.2 µM and 1.5 × 10(-2) µM(-1)sec(-1), and for 2,2',3-trihydroxybiphenyl, they were 168.0 µM and 0.5 × 10(-2) µM(-1)sec(-1), respectively. A phylogenetic tree of the large and small subunits of type II extradiol dioxygenases suggested that FlnD1D2 constitutes a novel subgroup among heterooligomeric type II extradiol dioxygenases.

Nanopore sequencing in distinguishing between wild-type and vaccine strains of Varicella-Zoster virus.

BACKGROUND: The introduction of varicella vaccines into routine pediatric immunization programs has led to a considerable reduction in varicella incidence. However, there have been reports of varicella, herpes zoster, and meningitis caused by the vaccine strain of varicella-zoster virus (VZV), raising concerns. Establishing the relationship between the wild-type and vaccine strains in VZV infections among previously vaccinated individuals is crucial. Differences in the single nucleotide polymorphisms (SNPs) among vaccine strains can be utilized to identify the strain. In this study, we employed nanopore sequencing to identify VZV strains and analyzed clinical samples. METHODS: We retrospectively examined vesicle and cerebrospinal fluid samples from patients with VZV infections. One sample each of the wild-type and vaccine strains, previously identified using allelic discrimination real-time PCR and direct sequencing, served as controls. Ten samples with undetermined VZV strains were included. After DNA extraction, a long PCR targeting the VZV ORF62 region was executed. Nanopore sequencing identified SNPs, allowing discrimination between the vaccine and wild-type strains. RESULTS: Nanopore sequencing confirmed SNPs at previously reported sites (105,705, 106,262, 107,136, and 107,252), aiding in distinguishing between wild-type and vaccine strains. Among the ten unknown samples, nine were characterized as wild strains and one as a vaccine strain. Even in samples with low VZV DNA levels, nanopore sequencing was effective in strain identification. CONCLUSION: This study validates that nanopore sequencing is a reliable method for differentiating between the wild-type and vaccine strains of VZV. Its ability to produce long-read sequences is remarkable, allowing simultaneous confirmation of known SNPs and the detection of new mutations. Nanopore sequencing can serve as a valuable tool for the swift and precise identification of wild-type and vaccine strains and has potential applications in future VZV surveillance.

Co-operative effects of thoracic X-ray irradiation and N-nitrosobis(2-hydroxypropyl) amine administration on lung tumorigenesis in neonatal, juvenile and adult Wistar rats.

Assessment of risks associated with childhood exposure to ionizing radiation when combined with chemical carcinogens is of great importance. We studied the age-dependence of the effect of combined exposure to ionizing radiation (IR) and a chemical carcinogen on lung carcinogenesis. Female 1-, 5-, and 22-week-old Wistar rats were locally irradiated on the thorax with X-rays (3.18 Gy) and/or were injected intraperitoneally with N-nitrosobis(2-hydroxypropyl)amine (BHP) (1g/kg body weight) 1 week after X-ray exposure or at 23 weeks of age. Rats were terminated at 90 weeks of age. We found that: (i) the incidence of lung tumors (adenoma and adenocarcinoma) increased slightly as a function of age at X-ray exposure, although this was not statistically significant, while the incidence induced by BHP decreased with increasing age at administration; (ii) combined exposure to X-rays at 5 or 22 weeks with BHP 1 week later enhanced the tumor incidence, and the effect at early-life stage (5 weeks irradiation) was more effective than that at late-life stage (22 weeks irradiation); (iii) combined exposure preferentially enhanced malignant transformation; (iv) although a longer interval between the X-ray and BHP treatments reduced the combined effect, risks of early-life irradiation at 1 or 5 weeks of age lasted into adulthood; (v) adenomas and adenocarcinomas induced by X-ray and/or BHP originated from surfactant apoprotein A-positive alveolar type II cells; and (vi), extracellular signal-regulated kinase pathway activation was observed in half the adenocarcinomas, regardless of the exposure schedule. In conclusion, combined exposure may enhance lung tumorigenesis more synergistically at early-life stage (5 weeks of age) than later-life stage.

Oxidative stress is an essential factor for the induction of anhydrobiosis in the desiccation-tolerant midge, Polypedilum vanderplanki (Diptera, Chironomidae).

The sleeping chironomid (Polypedilum vanderplanki) is the only insect capable of surviving complete desiccation in an ametabolic state called anhydrobiosis. Here, we focused on the role of oxidative stress and we observed the production of reactive oxygen species (ROS) in desiccating larvae and in those exposed to salinity stress. Oxidative stress occurs to some extent in desiccating larvae, inducing carbonylation of proteins. Oxidative stress overcomes the antioxidant defenses of the larvae during the first hour following rehydration of anhydrobiotic larvae. It facilitates the oxidation of DNA and cell membrane lipids; however, these damages are quickly repaired after a few hours. In addition to its deleterious effects, we demonstrated that artificial exposure to oxidative stress could induce a response similar to desiccation stress, at the transcriptome and protein levels. Furthermore, the response of anhydrobiosis-related genes to desiccation and salinity stress was inhibited by antioxidant treatment. Thus, we conclude that oxidative stress is an essential trigger for inducing the expression of protective genes during the onset of anhydrobiosis in desiccating of P. vanderplanki larvae.

Proteomic Analysis Reveals Novel Plasma Biomarkers for Neurological Complications in Patients With Congenital Cytomegalovirus Infection.

BACKGROUND: Congenital cytomegalovirus (cCMV) infection is a leading cause of nonhereditary neurological complications. When considering antiviral treatment, it is important to differentiate between symptomatic and asymptomatic patients. This study aimed to identify candidate plasma biomarkers for neurological complications of cCMV infection using proteomic analysis. METHODS: This study retrospectively enrolled five patients with symptomatic cCMV infection, four with asymptomatic cCMV infection with isolated sensorineural hearing loss (SNHL), and five with asymptomatic cCMV infection. The plasma samples were collected during neonatal period. The peptides were analyzed using liquid chromatography-mass spectrometry. The concentrations of differentially expressed proteins were validated using an enzyme-linked immunosorbent assay. RESULTS: A total of 456 proteins were identified and quantified. The levels of 80 proteins were significantly different between patients with and without cCMV-related symptoms including isolated SNHL. The levels of 31 proteins were significantly different between patients with and without neuroimaging abnormalities. The plasma concentrations of Fms-related receptor tyrosine kinase 4 in patients with cCMV-related symptoms were significantly higher than those in patients with asymptomatic cCMV infection. Moreover, plasma peptidylprolyl isomerase A levels were significantly higher in patients with neuroimaging abnormalities than in those without. CONCLUSIONS: Proteomic analysis of patients with cCMV infection showed that Fms-related receptor tyrosine kinase 4 and peptidylprolyl isomerase A could be novel diagnostic biomarkers for neurological complications of cCMV infection.

Lung-Cancer Risk in Mice after Exposure to Gamma Rays, Carbon Ions or Neutrons: Egfr Pathway Activation and Frequent Nuclear Abnormality.

Lung is one of the high-risk organs for radiation-induced carcinogenesis, but the risk of secondary lung-cancer development after particle-beam therapy and the underlying mechanism(s) remain to be elucidated. To investigate the effects of particle-beam radiation on adjacent normal tissues during cancer therapy, 7-week-old male and female B6C3F1 mice were irradiated with 0.2-4 Gy of gamma rays (for comparison), carbon ions (290 MeV/u, linear energy transfer 13 keV/µm), or fast neutrons (0.05-1 Gy, mean energy, ∼2 MeV), and lung-tumor development was assessed by histopathology. Mice irradiated with ≥2 Gy of carbon ions or ≥0.2 Gy of neutrons developed lung adenocarcinoma (AC) significantly sooner than did non-irradiated mice. The relative biological effectiveness values for carbon ions for lung AC development were 1.07 for male mice and 2.59 for females, and the corresponding values for neutrons were 4.63 and 4.57. Genomic analysis of lung ACs revealed alterations in genes involved in Egfr signaling. Hyperphosphorylation of Erk and a frequent nuclear abnormality (i.e., nuclear groove) were observed in lung ACs of mice irradiated with carbon ions or neutrons compared with ACs from non-irradiated or gamma-ray-irradiated groups. Our data indicate that the induction of lung AC by carbon ions occurred at a rate similar to that for gamma rays in males and approximately 2-to 3-fold greater than that for gamma rays in females. In contrast, the effect of neutrons on lung AC development was approximately 4- to 5-fold greater than that of gamma rays. Our results provide valuable information concerning risk assessment of radiation-induced lung tumors after particle-beam therapy and increase our understanding of the molecular basis of tumor development.

Genetic characterisation of genes involved in the upper pathway of carbazole metabolism from the putative Kordiimonas sp.

The car genes from a carbazole (CAR)-degrading bacterium, Kordiimonas sp. OC9, were functionally and transcriptionally analysed. The enzymatic activity for the protein coded by carBaBb using pBOC93 (carAaAcBa), pBOC93-2 (carAaAcBb), and pBOC94 (carAaAcBaBb) was confirmed. Resting cells using Escherichia coli harbouring pBOC95 (carAaAcBaBbC) revealed the function of the carC gene product in the conversion of CAR to anthranilic acid by expressing it with CarAaAcBaBb. The pathway of CAR metabolism to anthranilic acid in marine CAR-degraders was elucidated. Transcriptional analysis using RT-PCR revealed that car genes are related to CAR degradation in response to CAR exposure in strain OC9. RT-PCR analysis of the operon structure showed that the car gene cluster of strain OC9 has two distinct operons in one car gene cluster. The localisation of the car gene cluster of strain OC9 was also determined.

RGB-color forward-viewing spectrally encoded endoscope using three orders of diffraction.

Spectrally encoded endoscopy (SEE) is an ultra-miniature endoscopy technology that encodes each spatial location on the sample with a different wavelength. One challenge in SEE is achieving color imaging with a small probe. We present a novel SEE probe that is capable of conducting real-time RGB imaging using three diffraction orders (6th order diffraction of the blue spectrum, 5th of green, and 4th of red). The probe was comprised of rotating 0.5 mm-diameter illumination optics inside a static, 1.2 mm-diameter flexible sheath with a rigid distal length of 5 mm containing detection fibers. A color chart, resolution target, and swine tissue were imaged. The device achieved 44k/59k/23k effective pixels per R/G/B channels over a 58° angular field and differentiated a wide gamut of colors.

Cells from an anhydrobiotic chironomid survive almost complete desiccation.

Dry-preservation of nucleated cells from multicellular animals represents a significant challenge in life science. As anhydrobionts can tolerate a desiccated state, their cells and organs are expected to show high desiccation tolerance in vitro. In the present study, we established cell lines derived from embryonic tissues of an anhydrobiotic chironomid, Polypedilum vanderplanki, designated as Pv11 and Pv210. Salinity stress induced the expression of a set of anhydrobiosis-related genes in both Pv11 and Pv210 cells, suggesting that at least a part of cells can autonomously control the physiological changes for the entry into anhydrobiosis. When desiccated with medium supplemented with 300 mM trehalose or sucrose and stored for 4 weeks in dry air (approximately 5% relative humidity), a small percentage of the cells was found to be viable upon rehydration, although surviving cells seemed not to be able to multiply. We also attempted dry-preservation of organs isolated from P. vanderplanki larvae, and found that a proportion of cells in some organs, including fat body, testis, nerve and dorsal vessel, tolerated in vitro desiccation.

Cloning and nucleotide sequences of carbazole degradation genes from marine bacterium Neptuniibacter sp. strain CAR-SF.

The marine bacterium Neptuniibacter sp. strain CAR-SF utilizes carbazole as its sole carbon and nitrogen sources. Two sets of clustered genes related to carbazole degradation, the upper and lower pathways, were obtained. The marine bacterium genes responsible for the upper carbazole degradation pathway, carAa, carBa, carBb, and carC, encode the terminal oxygenase component of carbazole 1,9a-dioxygenase, the small and large subunits of the meta-cleavage enzyme, and the meta-cleavage compound hydrolase, respectively. The genes involved in the lower degradation pathway encode the anthranilate dioxygenase large and small subunit AntA and AntB, anthranilate dioxygenase reductase AntC, 4-oxalocrotonate tautomerase, and catechol 2,3-dioxygenase. Reverse transcription-polymerase chain reaction confirmed the involvement of the isolated genes in carbazole degradation. Escherichia coli cells transformed with the CarAa of strain CAR-SF required ferredoxin and ferredoxin reductase for biotransformation of carbazole. Although carAc, which encodes the ferredoxin component of carbazole 1,9a-dioxygenase, was not found immediately downstream of carAaBaBbC, the carAc-like gene may be located elsewhere based on Southern hybridization. This is the first report of genes involved in carbazole degradation isolated from a marine bacterium.

Isolation and characterization of the gene encoding the chloroplast-type ferredoxin component of carbazole 1,9a-dioxygenase from a putative Kordiimonas sp.

Carbazole (CAR)-degrading genes (carRAaCBaBb) were isolated from marine CAR-degrading isolate strain OC9 (probably Kordiimonas gwangyangensis) using shotgun cloning experiments and showed 35-65% similarity with previously reported CAR-degrading genes. In addition, a ferredoxin-like gene (carAc) was found downstream of carR, although it was not homologous with any reported ferredoxin components of the CAR 1,9a-dioxygenase (CARDO) system. The carAc-deduced amino acid sequence possessed consensus sequences for chloroplast-type iron-sulfur proteins for binding the [2Fe-2S] cluster. These car genes were arranged in the order of carAcRAaCBaBb, but carRAc and carAaCBaBb genes were the opposite orientation. Escherichia coli JM109 cells harboring pBOC91 (carAa) converted CAR to 2'-aminobiphenyl-2,3-diol at a ratio of 12%, and the transformation ratio of CAR increased from 12 to 100% when carAc was added, indicating that CarAc is the ferredoxin component of the CARDO system in strain OC9. This is the first finding of a chloroplast-type ferredoxin component in a CARDO system. Biotransformation tests with aromatic compounds revealed that the strain OC9 CarAaAc showed activity with polycyclic aromatic hydrocarbons and dioxin compounds and exhibited significant activity for fluorene, unlike previously reported CARDOs.

High hydrostatic pressure tolerance of four different anhydrobiotic animal species.

High hydrostatic pressure (HHP) can induce physical changes in DNA, proteins, and lipids, causing lethal or sublethal damage to organisms. However, HHP tolerance of animals has not been studied sufficiently. In this study, HHP tolerance of four species of invertebrate anhydrobiotes (the tardigrade Milnesium tardigradum, a nematode species in the family Plectidae, larvae of Polypedilum vanderplanki, and cysts of Artemia franciscana), which have the potential to enter anhydrobiosis upon desiccation, were investigated by exposing them to 1.2 GPa for 20 minutes. This exposure killed the anhydrobiotes in their ordinary hydrated state, but did not affect their survival in the anhydrobiotic state. The results indicated that the hydrated anhydrobiotes were vulnerable to HHP, but that HHP of 1.2 GPa was not sufficient to kill them in anhyrdobiosis.

Isolation and characterization of a car gene cluster from the naphthalene, phenanthrene, and carbazole-degrading marine isolate Lysobacter sp. strain OC7.

The novel carbazole (CAR)-degrading bacterium Lysobacter sp. strain OC7 has been isolated from seawater and can also utilize naphthalene and phenanthrene as its sole carbon and energy source. The CAR-degradative gene cluster was isolated and encoded five complete open reading frames (ORFs) and two truncated ORFs. Among them, four ORFs showed 40-50% similarity with previously reported CAR-degradative genes. Ferredoxin (carAc) and ferredoxin reductase (carAd) genes, which are necessary for the CAR 1,9a-dioxygenase system, were not found in this car gene cluster. The car (OC7) gene transcripts were strongly detected when CAR was provided. However, these transcripts were also detected when naphthalene was provided. The resting cell reaction with Escherichia coli revealed that CarAa(OC7) can use CarAc and CarAd of Pseudomonas resinovorans CA10 as ferredoxin and ferredoxin reductase, respectively, and converted CAR to 2'-aminobiphenyl-2,3-diol. In 13 marine CAR-degrading isolates, only Caulobacter sp. strain OC6 hybridized with the car (OC7) gene cluster probe. This is the first report showing CAR-degradative genes from the genus Lysobacter.

Functional analysis of the thermophilic denitrifying bacterium Geobacillus sp. strain TDN01 in continuous culture.

The thermophilic denitrifying bacterium Geobacillus sp. strain TDN01 was examined to determine the effects of nitrogen and carbon sources and nitrate and nitrite concentrations on denitrification in a batch culture. The specific nitrate removal rate was 12 times higher with ammonia than without ammonia. The consumption rates of nitrate and succinate were proportional. Furthermore, the growth rates with 120 and 150 mM nitrate were only slightly lower than those with 60 mM and did not cause notable growth inhibition. Denitrification ability in continuous culture was analyzed based on the data for batch culture. The maximum specific growth rate micromax and substrate saturation constant KS in the Monod equation were determined by gradually changing the dilution rate. The maximum denitrification rate was six times higher than that of mesophilic bacteria.

Cultivation characteristics of denitrification by thermophilic Geobacillus sp. strain TDN01.

Nine thermophilic denitrification bacteria were isolated from field soil, mud, and spa samples. The alignment of 16S rDNA showed that all were identical to the genus Geobacillus. Two of the bacteria produced N2O and N2 gas and the other seven strains produced N2 gas from nitrate. We examined the growth substrates for Geobacillus TDN01 and determined that sodium succinate, pyruvate, formate, acetate, glycerol, glucose, sucrose, and cellobiose well supported growth of the isolate. Growth occurred under the following concentration of NO3- and phosphate: 10-60 mmol/L, and 0.1-50 mmol/L, respectively. Thermophilic TDN01 grown on sodium succinate accumulated nitrite. A time course of denitrification by Geobacillus TDN01 in a jar fermentor revealed that maintaining a pH of around 7 is important for denitrification without accumulating NO2. The NO3- and NO2- consumption ratios of Geobacillus were 44-75 and 9-41 times higher, respectively, than those of Pseudomonas stutzeri JCM 5965T.

Production of human erythropoietin by chimeric chickens.

The use of transgenic avian allows cost effective and safe production of pharmaceutical proteins. Here, we report the successful production of chimeric chickens expressing human erythropoietin (hEpo) using a high-titer retroviral vector. The hEpo expressed by transgenic hens accumulated abundantly in egg white and had N- and O-linked carbohydrates. While attachment of terminal sialic acid and galactose was incomplete, portions of N- and O-linked carbohydrates were present. In vitro biological activity of egg white-hEpo was comparable to that produced by recombinant CHO cells.

Effects of dehydration rate on physiological responses and survival after rehydration in larvae of the anhydrobiotic chironomid.

Strategies to combat desiccation are critical for organisms living in arid and semi-arid areas. Larvae of the Australian chironomid Paraborniella tonnoiri resist desiccation by reducing water loss. In contrast, larvae of the African species Polypedilum vanderplanki can withstand almost complete dehydration, referred to as anhydrobiosis. For successful anhydrobiosis, the dehydration rate of P. vanderplanki larvae has to be controlled. Here, we desiccated larvae by exposing them to different drying regimes, each progressing from high to low relative humidity, and examined survival after rehydration. In larvae of P. vanderplanki, reactions following desiccation can be categorized as follows: (I) no recovery at all (direct death), (II) dying by unrepairable damages after rehydration (delayed death), and (III) full recovery (successful anhydrobiosis). Initial conditions of desiccation severely affected survival following rehydration, i.e. P. vanderplanki preferred 100% relative humidity where body water content decreased slightly. In subsequent conditions, unfavorable dehydration rate, such as more than 0.7 mg water lost per day, resulted in markedly decreased survival rate of rehydrated larvae. Slow dehydration may be required for the synthesis and distribution of essential molecules for anhydrobiosis. Larvae desiccated at or above maximum tolerable rates sometimes showed temporary recovery but died soon after.