Individuals also make significantly shorter journeys of less than

Individuals also make significantly shorter journeys of less than 5 weeks, and were more likely to visit the TAVC more than 30 days before departure than in the past. Only 24% of the Mecca travelers accepted the recommended dTP vaccine. Possible reasons for this low acceptance are that most of these

travelers do not come to our clinic for health advice, but for a vaccination that is necessary to obtain a visa. Other reasons can be the costs of the vaccinations, and that people are not informed about the possible risks and recommended vaccinations prior to their visit to us. Communication is often difficult because of language barriers. In univariate analysis, women, second-generation Muslims, and older people were significantly more likely to accept dTP vaccination than Buparlisib men and younger people. In multivariate analysis, the variable second-generation Muslims was no longer significant, and younger

see more people were significantly more likely to accept dTP. Schlagenhauf and colleagues also found that women are significantly more likely to obtain pretravel advice.6 Another predictor for dTP acceptance in our study is health. The more unhealthy people are, the more likely it is that they will accept the recommended vaccinations. Looking at the specific disorders, individuals with heart or vascular disorders, those with liver and gastrointestinal disorders, and those with other disorders were significantly more often likely to accept the dTP vaccine. Apparently, the more vulnerable people’s health, the more they are willing to protect themselves from other diseases. The reason that, independently, younger PAK5 people are more likely to accept recommended vaccinations is possibly because they are better informed, and communication is easier because

there are no language barriers. In conclusion, only a quarter of Mecca travelers who visit a travel clinic for their mandatory meningitis vaccination also take other, recommended, vaccinations. Women, younger people, and less healthy people are more likely to follow recommendations. To improve uptake, which in this scenario would be more people accepting recommended vaccinations, Islamic organizations that provide Mecca travelers with travel advice should be better informed, not only about the required vaccinations, but also about recommended vaccinations and other health advice. We thank Dr Lothar D.J. Kuijper, Vrije Universiteit Amsterdam, for his support of this study. The authors state they have no conflicts of interest to declare. “
“Travelers to countries where rabies is endemic may be at risk of rabies exposure. We assessed rabies immunization of travelers attending a travel clinic in Thailand. The medical charts of international travelers who came for preexposure (PrEP) or postexposure (PEP) rabies prophylaxis at the Queen Saovabha Memorial Institute (QSMI), Bangkok, Thailand between 2001 and 2011 were retrospectively reviewed.

qRT-PCR was preformed

qRT-PCR was preformed buy PLX3397 on the same samples used for microarray analysis using primer sets for eight genes (dnaK, espA, lpfD, macA, ompA, recA, stx1A, stx2A) to confirm significant transcriptional differences due to treatment. The Express One-Step SYBR GreenER kit (Invitrogen) was used for qRT-PCR with the Mx3005P QPCR System (Stratagene, La Jolla, CA) and mxpro 4.1 software. Reaction volume for each well totaled 15 μL and

contained 3.69 μL of water, 7.5 μL qRT-PCR mix, 1.2 μL of each primer (Table 1) at 2.5 μM, 0.03 μL ROX, 1 μL of sample RNA, and 0.375 μL (75 U) SuperScript III. Six biological replicates for each treatment were randomly chosen for qRT-PCR validation and were run in duplicate. Gene btuD was used as a reference gene because it demonstrated no detectable differential expression due www.selleckchem.com/products/Thiazovivin.html to treatment and had a

small variance on the microarrays. The method described in Gallup & Ackermann (2006) was used for primer optimization, detection of inhibition, and troubleshooting of qRT-PCR. A four-point standard curve was constructed with duplicate samples of a collection of all RNAs (Stock 1) and used for the calculation of efficiencies for target genes and the reference gene (Gallup & Ackermann, 2006). The ISU equation was used to calculate fold change between treatment and control samples (Gallup & Ackermann, 2006), and the Student’s t-test was used to determine significance of differences. Confidence threshold values that were greater than 2 SD from the mean were considered outliers and were ZD1839 clinical trial not used in data analysis. The microarray dataset can be accessed from the National Center for Biological Informatics Gene Expression Omnibus using Series accession number GSE16762 (http://www.ncbi.nlm.nih.gov/geo/). Initially, we determined the survivability of E. coli O157:H7 in A. castellanii under the conditions of the microarray study (Fig. 1).

Initial CFUs of E. coli O157:H7 began at 109 and fell 5 logs during the first 2 h before leveling off to 103–104 for the next 14 h (Fig. 1). The addition of gentamicin to the culture media after a 30-min ingestion period did not affect the viability of A. castellanii or the bacteria within (data not shown). Microarrays were used to compare steady-state transcript levels of E. coli O157:H7 within A. castellanii to planktonic cultures to determine the effect of the intracellular environment. Based on the data from the internal survival curve, an incubation period of 4.5 h was chosen for the microarray study. This included an initial 30 min for A. castellanii engulfment of E. coli, 2 h for killing extracellular bacteria with gentamicin, and an additional 2 h for transcriptional activity to stabilize and allow dead bacteria to be degraded. All RNA preparations fulfilled our criteria for integrity and purity and lacked contamination with A.

qRT-PCR was preformed

qRT-PCR was preformed Selleck FK228 on the same samples used for microarray analysis using primer sets for eight genes (dnaK, espA, lpfD, macA, ompA, recA, stx1A, stx2A) to confirm significant transcriptional differences due to treatment. The Express One-Step SYBR GreenER kit (Invitrogen) was used for qRT-PCR with the Mx3005P QPCR System (Stratagene, La Jolla, CA) and mxpro 4.1 software. Reaction volume for each well totaled 15 μL and

contained 3.69 μL of water, 7.5 μL qRT-PCR mix, 1.2 μL of each primer (Table 1) at 2.5 μM, 0.03 μL ROX, 1 μL of sample RNA, and 0.375 μL (75 U) SuperScript III. Six biological replicates for each treatment were randomly chosen for qRT-PCR validation and were run in duplicate. Gene btuD was used as a reference gene because it demonstrated no detectable differential expression due selleck inhibitor to treatment and had a

small variance on the microarrays. The method described in Gallup & Ackermann (2006) was used for primer optimization, detection of inhibition, and troubleshooting of qRT-PCR. A four-point standard curve was constructed with duplicate samples of a collection of all RNAs (Stock 1) and used for the calculation of efficiencies for target genes and the reference gene (Gallup & Ackermann, 2006). The ISU equation was used to calculate fold change between treatment and control samples (Gallup & Ackermann, 2006), and the Student’s t-test was used to determine significance of differences. Confidence threshold values that were greater than 2 SD from the mean were considered outliers and were tuclazepam not used in data analysis. The microarray dataset can be accessed from the National Center for Biological Informatics Gene Expression Omnibus using Series accession number GSE16762 (http://www.ncbi.nlm.nih.gov/geo/). Initially, we determined the survivability of E. coli O157:H7 in A. castellanii under the conditions of the microarray study (Fig. 1).

Initial CFUs of E. coli O157:H7 began at 109 and fell 5 logs during the first 2 h before leveling off to 103–104 for the next 14 h (Fig. 1). The addition of gentamicin to the culture media after a 30-min ingestion period did not affect the viability of A. castellanii or the bacteria within (data not shown). Microarrays were used to compare steady-state transcript levels of E. coli O157:H7 within A. castellanii to planktonic cultures to determine the effect of the intracellular environment. Based on the data from the internal survival curve, an incubation period of 4.5 h was chosen for the microarray study. This included an initial 30 min for A. castellanii engulfment of E. coli, 2 h for killing extracellular bacteria with gentamicin, and an additional 2 h for transcriptional activity to stabilize and allow dead bacteria to be degraded. All RNA preparations fulfilled our criteria for integrity and purity and lacked contamination with A.

Methods  Fifty teeth from 37 healthy children aged 3–8 years wit

Methods.  Fifty teeth from 37 healthy children aged 3–8 years with pulpally involved primary molars needing root canal procedures were treated with 3Mix or selleckchem Vitapex®

before restoration with stainless steel crowns. The research employed a prospective single-blinded randomized design. The subjects were followed up clinically and radiographically at 6 and 12 months, respectively. The outcome was compared using a Z-test with a significance level of 0.05. Results.  Both groups showed 100% and 96% clinical success at 6 and 12 months, respectively. At 6 months, radiographic success of 3Mix and Vitapex® was 84% and 80%, respectively, and at 12 months, radiographic success of 3Mix and Vitapex® was 76% and 56%, respectively. Considering the radiographic findings at the end of 6 and 12 months, no statistically significant differences were found between the two groups (P = 0.356 and 0.068, respectively).

Conclusion.  3Mix and Vitapex® can be used as a root canal treatment agent in pulpally involved primary teeth. “
“International Journal of Paediatric Dentistry 2011; 22: 37–43 Objectives.  To evaluate the reliability of panoramic radiographs (PRs) for identifying supernumerary teeth (ST) and to determine whether the level of dental training of the observer influenced the identification of ST. Methods.  Seventy-five PRs were randomly selected from the patient records and 18 examiners independently rated 25 radiographs each, for specific risk factors as well as for a measure of adequacy. Subsequently, the results were paired with those of the other examiners who assessed the find more same set of PRs. Descriptive statistics were computed using Fisher’s exact test,

and kappa statistics were used to assess MRIP the inter- and intra-observer reliability. Results.  Four hundred and fifty PRs were available for analysis. The overall sensitivity and specificity figures were 50% and 98.3%, whereas the positive and negative predictive values were 90.6% and 83.6%, respectively. The sensitivity figures for Junior House Dental Officers and Postgraduate Paediatric Dental Trainees were 39.2% and 60.8%, whereas the specificity figures were 99.4% and 95% with slight inter-examiner and moderate intra-examiner reliability. Conclusions.  Panoramic radiographs are unreliable for identifying ST, and higher level of dental training is essential for identifying ST. “
“International Journal of Paediatric Dentistry 2010; 20: 173–178 Background.  Children with previous experience of infective endocarditis or with prosthetic heart valve are considered at very high risk for infective endocarditis. Aim.  The aim of this study was to compare the dental health of a group of these children with a group of healthy controls and to determine parental awareness of the importance of good oral health. Design.  Oral examination was carried out in 28 children with previous infective endocarditis or a prosthetic heart valve to assess oral health.

An annual offer of a full sexual health screen (regardless of rep

An annual offer of a full sexual health screen (regardless of reported history) and the outcome documented in the HIV case notes, including whether declined (IIb). Syphilis serology should be documented at baseline and performed yearly. In individuals or groups at increased risk of syphilis (MSM), syphilis serology OSI-906 should be considered with routine HIV follow-up (2–4 times yearly) (IIb). All women should have cervical smears

performed annually (IV). Screening for anal dysplasia by anal cytology may be beneficial; however, there is insufficient evidence at this time to support its routine introduction (IV). Gender-specific aspects of HIV monitoring will be discussed fully in the BHIVA women’s guidelines currently under

development. Approximately 20% of HIV-infected individuals accessing care in the UK are aged 50 years or more [1]. The prevalence of ageing HIV-infected PI3K inhibitor individuals continues to increase as a result of: (i) greater survival rates among HIV-infected patients; (ii) delayed recognition of the infection in older individuals; and (iii) continued new infections in older individuals. There is a need to adapt the management of HIV-infected individuals to ensure that the clinical needs of these individuals continue to be met as they age. However, very little is known about the likely healthcare needs of these patients. Existing reports on the clinical picture of HIV infection among older individuals are largely anecdotal; HIV may accelerate several

age-related conditions, and HIV-infected individuals may experience accelerated frailty, accelerated bone mineral loss and different levels of drug absorption and metabolism compared with their younger counterparts. Impaired glomerular function, impaired tubular function and proteinuria are all more common in the elderly. While this age-related decline in renal function is unlikely to result in severe kidney failure, it may affect many homeostatic processes, which may have implications for exacerbation of bone mineral loss and/or increased cardiovascular risk. The impact on adherence and potential drug–drug interactions of treatment for age-related comorbidities in patients who may be receiving ART has not been documented. HIV infection AZD9291 order and ageing are also both associated with changes in immunity and host defence. The potential for full immune restoration among older individuals receiving HAART for prolonged periods of time has not been fully investigated. In older individuals, drug pharmacokinetics (absorption, distribution, metabolism, and elimination) are altered [2] as a result of: (i) changes in gastric pH; (ii) body fat increase and water decrease; (iii) reductions in liver volume, blood flow and metabolic enzyme activity; (iv) decreased renal function.

The risk of death from each specific cause was higher in IDUs tha

The risk of death from each specific cause was higher in IDUs than non-IDUs, with particularly marked increases in risk for liver-related deaths, and those from violence and non-AIDS infection. While liver-related deaths and deaths from direct effects of substance abuse appear to explain much of the excess mortality in IDUs, they are at increased risk for many other causes of death, which may relate to suboptimal management of HIV disease in these

individuals. Injecting drug use (IDU) is one of the most frequent routes of HIV transmission in FG-4592 clinical trial many industrialized countries [1] and is responsible for up to one-third of HIV transmission globally, outside of sub-Saharan Africa [2]. Since the introduction of combination antiretroviral therapy (cART) in 1996, mortality rates related to HIV infection have significantly decreased [3–9]. Rates of morbidity and mortality subsequent to initiation of cART are higher in HIV-positive IDUs than in other HIV-positive persons [10–13], although some studies found only

limited evidence for this effect [6,14,15]. Several factors may contribute to the relatively poor response to treatment observed in HIV-positive patients who have a history of IDU. They have been shown to have decreased access to HIV care and treatment [16,17], more comorbid conditions associated with drug use and addiction [such as hepatitis C virus (HCV) coinfection], poorer adherence to treatment [18], and more adverse drug interactions [19,20]. They are also more likely to come from MEK inhibitor particular ethnic or racial groups that have historically been disadvantaged with respect

to health outcomes [21]. In some studies, immunological or virological responses to cART appeared to be lower in HIV-positive IDUs than in other patients [11,22]. However, it is important to distinguish between those who are and are not actively injecting G protein-coupled receptor kinase drugs, as the former will have additional risks from overdose, accidents and violence. Given the high prevalence of IDU among HIV-positive individuals receiving cART, it is important to understand what factors affect disease progression and death in this group: for example, in order to design programmes to reduce disparities in health outcomes between IDUs and non-IDUs receiving cART. We examined determinants of disease progression and death among IDUs and non-IDUs initiating cART in participants in a large multinational collaboration of HIV treatment programmes, and compared causes of death in IDU and non-IDU populations. The Antiretroviral Therapy Cohort Collaboration (ART-CC) is a multinational collaboration of HIV cohort studies. The collaboration has been described in detail elsewhere [12,23,24]. In brief, it was established in 2001, updated in 2004, 2006 and 2008, and includes cohort studies from Canada, Europe and the USA.

DNA sequence analysis of three clones indicates that the compleme

DNA sequence analysis of three clones indicates that the complementing genes are homologous to, but substantially different from, Lapatinib concentration known polyhydroxyalkanaote synthase-encoding genes. Thus we have demonstrated the ability to isolate diverse genes for polyhydroxyalkanaote synthesis

by functional complementation of defined mutants. Such genes might be of use in the engineering of more efficient systems for the industrial production of bioplastics. The use of functional complementation will also provide a vehicle to probe the genetics of polyhydroxyalkanaote metabolism and its relation to carbon availability in complex microbial assemblages. Petrochemically derived plastics are extremely useful materials, and they dominate many sectors of the industrial economy. Alpelisib in vivo However, they are inherently costly to the environment. They are produced from nonrenewable fossil fuels, their waste accumulates due to their recalcitrance to biodegradation, and their production cost will likely escalate as oil reserves are depleted. There is much interest in developing viable alternatives to these plastics. Polyhydroxyalkanoates (PHA) are commonly accumulated bacterial intracellular carbon storage polymers (Steinbüchel & Lütke-Eversloh, 2003; Trainer & Charles, 2006; Keshavarz & Roy, 2010). Their function

is to guard against stresses at the level of nutritional carbon and energy balance. Genetic studies of polyhydroxyalkanaote synthesis have been carried out in several bacteria. The central enzyme, polyhydroxyalkanaote

synthase encoded by phaC, catalyses the polymerization of hydroxyacyl-CoA molecules, driven by the energy released from CoA hydrolysis. These polymers are arranged in the cell as inert granules, complexed with associated proteins. Upon starvation or other stress, they can be depolymerized Histamine H2 receptor to provide a source of carbon and energy to sustain the cell. They are thus of central importance to the metabolic functioning of many bacteria. While the most common polyhydroxyalkanaote is poly-3-hydroxybutyrate (PHB), the diversity of polyhydroxyalkanaote is significant, with over 150 different possible monomeric constituents present in different combinations within a given polymer (Steinbüchel & Lütke-Eversloh, 2003). This structural diversity is reflected in the wide range of physical properties demonstrated by these polymers. Polyhydroxyalkanaote polymers are being developed for industrial purposes, as biodegradable replacements for fossil-fuel derived plastics, and as materials with unique properties. Major research efforts are focused on developing the ability to produce these materials in an economically competitive manner so that they will be commercially viable. Polyhydroxyalkanaote’s structure is determined in part by polyhydroxyalkanaote synthase’s substrate specificity, and there is considerable interest in determining the basis for such substrate specificity.

A second result was obtained

A second result was obtained Cabozantinib nmr by using SR95531 at concentrations sufficiently high to rapidly block the tonic current above the chloride equilibrium potential (ECl). Surprisingly, below ECl, SR95531 (10–40 μm) activated a sustained inward current, associated with a conductance increase, and resistant to bicuculline or PTX (100 μm). Similarly, after blockade of the bicuculline-sensitive current, SR95531 activated an

outward current above ECl. The bicuculline-resistant anionic current activated by SR95531 could be blocked by a GABAC receptor antagonist. Thus, two types of inhibitory GABA receptors, belonging to the GABAA and GABAC families, are able to show a sustained activity in HMs and provide promising targets for neuroprotection

under overexcitatory situations known to easily damage these particularly fragile neurons. “
“Food restriction has been reported to have positive effects on cognition. This study examines how another environmental selleck inhibitor factor, daylength, can alter the impact of food restriction on the brain and behavior. Female California mice (Peromyscus californicus), housed on either long days (16 h of light and 8 h of darkness) or short days (8 h of light and 16 h of darkness), were restricted to 80% of their normal baseline food intake or provided with food ad libitum. Testing in a Barnes maze revealed that the effects of food restriction depended on photoperiod, and that these effects differed for acquisition vs. reversal learning. During acquisition testing, food restriction increased latency to finding the target hole in short-day mice but not in long-day mice. In reversal

testing, food restriction decreased latency to finding the target hole in long-day Casein kinase 1 mice but not in short-day mice. Latency to finding the hole was positively and independently correlated with both errors and time spent freezing, suggesting that changes in both spatial learning and anxiety-like behavior contributed to performance. Short days increased hippocampal expression of the synaptic protein, synapsin I, which was reversed by food restriction. Short days also reduced plasma corticosterone levels, but diet had no effect. There was no effect of diet or photoperiod on hippocampal expression of the glial marker, glial fibrillary acidic protein. The present findings suggest that, in female California mice, the differential effects of food restriction on acquisition and reversal learning are photoperiod-dependent. These results justify further testing of the relationship between food restriction and hippocampal synapsin I in the context of spatial learning. “
“The lateral habenula (LHb) is an epithalamic region with a crucial role in the regulation of midbrain monoaminergic systems. Over the past few years a renewed interest in the LHb has emerged due to studies highlighting its central role in encoding rewarding and aversive aspects of stimuli.

volcanii and E coli pAJ successfully expressed proteins in Hfx

volcanii and E. coli. pAJ successfully expressed proteins in Hfx. volcanii or E. coli, rendering it feasible to express target proteins in corresponding domains. In addition, pAJ contains a multiple cloning site with 11 restriction sites and a 6×His tag sequence, and the vector size was decreased to 8903 bp. To the best of our knowledge, pAJ is the first reported shuttle expression vector that can express proteins in both Bacteria and Archaea. Importantly, pAJ can even express the haloarchaeal heat shock click here protein DnaK in both domains. In conclusion, this novel vector only provides researchers with a new means to manipulate genes

or express proteins in Haloarchaea but also serves as a convenient tool for the comparative study of the function of some highly conserved genes in Haloarchaea and in Bacteria. “
“The present study describes the assimilation of phenanthrene by an aerobic bacterium, Ochrobactrum sp. strain PWTJD, isolated from municipal waste-contaminated soil sample

utilizing phenanthrene as a sole source of carbon and energy. The isolate was identified as Ochrobactrum sp. based on the morphological, nutritional and biochemical characteristics as well as 16S rRNA gene sequence analysis. A combination of chromatographic analyses, oxygen uptake assay and enzymatic studies confirmed the degradation of phenanthrene by the strain PWTJD via 2-hydroxy-1-naphthoic acid, salicylic acid and catechol. The strain PWTJD could also utilize 2-hydroxy-1-naphthoic acid and MEK inhibitor salicylic acid, while the former was metabolized by a ferric-dependent meta-cleavage dioxygenase. In the lower pathway, salicylic acid was metabolized to catechol and was further degraded by catechol 2,3-dioxygenase to 2-hydroxymuconoaldehyde acid, ultimately leading to tricarboxylic acid cycle intermediates. This is the first report of

the complete degradation of a polycyclic aromatic hydrocarbon molecule by Gram-negative Ochrobactrum sp. describing the involvement of the meta-cleavage pathway of 2-hydroxy-1-naphthoic acid in phenanthrene assimilation. Polycyclic aromatic hydrocarbons (PAHs) comprise a large science and diverse group of priority environmental pollutants, which are ubiquitous contaminants derived from both natural and anthropogenic activities. Their abundance in the environment is of great concern, because many of them have been shown to be toxic, mutagenic and/or carcinogenic in nature (Mastrangelo et al., 1996; Marston et al., 2001; Xue & Warshawsky, 2005). The stability, persistency and carcinogenic index of PAHs increase with an increase in the number of aromatic rings, structural angularity and hydrophobicity (Marston et al., 2001). Phenanthrene has often been used as a model compound to study the microbial metabolism of bay- and K-region-containing PAHs because its structural skeletons are found in many carcinogenic PAHs.

In the presence of PCA, PcaU acts as an activator for the transcr

In the presence of PCA, PcaU acts as an activator for the transcription of the pca operon (Gerischer et al., 1998; Trautwein & Gerischer, 2001). In contrast, the reports on IclR-type repressors involved in the regulation of catabolic genes for aromatic compounds are limited to HmgR of P. putida U (Arias-Barrau et al., 2004), CatR of Rhodococcus erythropolis CCM2595 (Veselý et al., 2007), and PraR of Paenibacillus sp. strain JJ-1b (Kasai et al., 2009), which negatively regulate the homogentisate pathway genes, the catechol ortho-cleavage pathway genes, and the PCA 2,3-cleavage pathway

genes, respectively. Among these IclR-type repressors, only the research of the HmgR showed the binding of this repressor to the operator. Here, we focused on the regulation of iphACBDR operon controlled

by an IclR-type repressor, IphR. This Apoptosis Compound Library clinical trial is the first report to determine the transcription start site of iph operon, binding region of IphR, and effector molecule of IphR. Comamonas sp. strain E6 and its SCH772984 manufacturer mutants, DEIR and DEIA (Fukuhara et al., 2010) were grown in Luria–Bertani (LB) medium or in 0.2× LB medium at 30 °C. When required, 50 mg of kanamycin/liter or 30 mg of chloramphenicol/liter were added to the media. Escherichia coli strains JM109 and BL21(DE3) were grown in LB medium at 37 °C. For cultures of E. coli cells carrying antibiotic resistance markers, the media were supplemented with 100 mg of ampicillin/liter or 25 mg of kanamycin/liter. A set of deletion plasmids of pZSH2 (Fukuhara et al., 2010), pZSM1, pZSP08, pZSN06, pZSNE530, pZSNE347, and pZSNE198, was constructed by deletion using restriction enzymes or a Kilosequence kit (Takara Bio Inc.). To construct pZ347, pZ284, pZ274, and pZ255, the DNA fragments amplified by PCR using specific primer pairs (Supporting Information, Table Quisqualic acid S1) and pKS24 (Fukuhara et al., 2010) as a template were cloned into a promoter probe vector pPR9TZ (Kamimura et al.,

2010). Nucleotide sequences of the insert fragments were determined by the dideoxy termination method using a CEQ2000XL genetic analysis system (Beckman Coulter Inc.) The lacZ reporter plasmids were introduced into cells of E6 and DEIA by the triparental mating procedure. Cells of E6 and DEIA harboring each reporter plasmid pre-grown in 0.2× LB medium containing chloramphenicol were inoculated into the same fresh medium to an absorbance at 600 nm of 0.2. After 90 min of incubation at 30 °C, 5 mM IPA was added, and the cultures were incubated for another 120 min. The cells were washed twice with 20 mM Tris-HCl (pH 8.0) and resuspended in the same buffer, and broken by ultrasonication. The supernatant was collected by centrifugation (19 000 g, 15 min, 4 °C) and used as a crude enzyme. The β-galactosidase activities were measured using 4-methylumbelliferyl-β-d-galactopyranoside (Kamimura et al., 2010). The protein concentration was determined by the Bradford method (Bradford, 1976).