On September 2013 an attack on the Westgate mall in Kenya led to a four day siege, resulting in 67 fatalities and 175 wounded. During the crisis, Twitter became a crucial channel of communication between the government, emergency responders and the public, facilitating the emergency management of the event. The objectives of this paper are to present the main activities, use patterns and lessons learned from the use of the social media in the crisis. Using TwitterMate, a system developed to collect, store and analyze tweets, the main hashtags generated by the crowd and specific Twitter accounts of individuals, emergency responders and NGOs, were followed throughout the four day siege. A total of 67,849 tweets were collected and analyzed. Four main categories of hashtags were identified: geographical locations, terror attack, social support and organizations. The abundance of Twitter accounts providing official information made it difficult to synchronize and follow the flow of information. Many organizations posted simultaneously, by their manager and by the organization itself. Creating situational awareness was facilitated by information tweeted by the public. Threat assessment was updated through the information posted on social media. Security breaches led to the relay of sensitive data. At times, misinformation was only corrected after two days. Social media offer an accessible, widely available means for a bi-directional flow of information between the public and the authorities. In the crisis, all emergency responders used and leveraged social media networks for communicating both with the public and among themselves. A standard operating procedure should be developed to enable multiple responders to monitor, synchronize and integrate their social media feeds during emergencies. This will lead to better utilization and optimization of social media resources during crises, providing clear guidelines for communications and a hierarchy for dispersing information to the public and among responding organizations. In recent years, the social media, and especially Twitter, have emerged as important, near real-time communication channels and technologies during emergency response. Social media consist of tools that enable open and online exchange of information through conversation, interaction and exchange of user generated content A recent study showed that Kenya is the second most active country in Africa on Twitter, surpassed only by South Africa The use of social media in emergencies, specifically Twitter and Facebook, is growing. The different social media channels have become an integral tool in the response of official authorities and the general public to emergency situations Sentiment analysis  has been defined as the task of finding the opinions of authors about specific organizations In September 2013, an attack on the Westgate shopping mall in Kenya led to a four day siege, resulting in 67 fatalities and about 175 wounded. Around noon on September 21, an unknown number of armed terrorists entered the Westgate shopping mall in Nairobi, Kenya and took control. During the following days, the Kenyan security forces tried to retake the mall using ground and air forces. Kenyan emergency authorities managing the crisis established a perimeter surrounding the mall and provided support and medical treatment to the injured and their families. The Al-Shabaab militant group took responsibility for the attack through one of their Twitter accounts.The Kenya Westgate Mall attack provides a unique opportunity to assess and understand how emergency organizations utilize social media to improve their responses to the event through content and activity analysis. This research aims to address three main hypotheses:Sentiment of emergency dispatches/EOC (Emergency Operations Center) tweets will be more positive than the sentiment of tweets made by the managers in the field.Use of social media during urban terror attacks leads to breaches of security.Emergency managers new to Twitter will use it as a basic, one-way, communications channel.This case study offers two significant contributions. The first is a unique opportunity to study an event that took place in Africa, an understudied region in the field of social media. The second is the limited geographic nature of this event (which occurred in a defined set of buildings), enabling an understanding of the contribution of social media in an ongoing emergency. Currently, there is not enough evidence for best practice when incorporating social media in emergency response. In this study we make progress in understanding crisis communication patterns mediated by social media. Learning lessons from this event concerning the use of social media to create situational awareness can facilitate the creation of standard operating procedures for first responders that will encompass information sharing, control and command operations, and risk communication.The Kenya Westgate mall attack began on September 21, 2013 and lasted four days until September 24, 2013. Between September 21 and September 25, 2013, structured monitoring, collection, storing and analysis of Twitter information was carried out.Data collection was performed using TwitterMate, a self-developed system specifically intended to collect tweets from Twitter. TwitterMate's architecture includes four main components- 1. A graphical user interface (GUI) where a user can define the basic Twitter search according to one or more usernames, hashtags and specific tweet IDs. TwitterMate can also collect the followers of specific Twitter users. Through the username search, TwitterMate is able to extract all tweets made by a specific Twitter account. The searches cover Twitter Search, REST and Stream APIs to collect tweets, and can run in an automatic or manual mode. 2, An Excel-based data store where all tweets are saved. Tweets are saved according their search parameters in different tabs, and are broken into 11 different information classifications. 3. A generic built-in API section that can operate and consume any natural language processing REST API calls i.e., Alchemy API. The API calls can be run on a specific tab in the Excel to analyze tweets. 4. In the Excel, further text analysis is conducted using a series of macros and formulas to extract hashtags and user accounts from the messages and clean tweets, running them through NLP analysis. The use of TwitterMate during a number of time intervals, as presented in http://www.alchemyapi.com/), a collection of natural language processing tools which provides multiple targeting options for sentiment analysis, including document-level, entity-level, and keyword-level directional, relational and user-specified sentiment analysis. In the present work, we used document-level (tweet level) sentiment analysis. Each tweet received a sentiment score: −1 (negative sentiment), 0  and 1 (positive sentiment), and a general classification of polarity . Alchemy API has previously been used for sentiment analysis tasks in different contexts, including tourism As mentioned, TwitterMate has a built-in feature utilizing Alchemy API (@username); i.e., a ‘mention’ https://*) to URL, and converted @username to AT_USER. Finally, we removed stop words as they usually have little meaning. Such preprocessing is common and standard procedure in sentiment analysis Twitter enables users to add a direct reference to a user in their tweets by inserting the user's account name  and all governmental accounts and emergency organizations' accounts that participated in the response were followed and collected continually during the crisis, between September 21 and September 25. The participants followed were representatives of the Kenyan government and emergency authorities, and are thus considered formal and valid agents of Kenya's administration. Accordingly, tweets published by these organizations were considered as valuable and with impact on the population. We used the collected data to understand the progression and use of Twitter by the emergency authorities and to identify lessons learned. Using Statistical Package for the Social Sciences (SPSS), 19 we conducted a chi-squared test on the sentiment scores of the emergency authorities and their managers.The collection was made during the hours of the day as it required manual interventions as new hashtags were created. All tweets made by the accounts mentioned in In order to avoid bias we used the AlchemyAPI to analyze only original tweets communicated by emergency organizations and their managers, while disregarding re-tweets.t (2004) = 1.76, p<0.05 (one-tailed), d′ = 0.12. Managers' average sentiment  was more positive than organizations' average sentiment . However, using a two-sided chi-squared statistical test we found no difference in sentiment expression between managers and organizations, although descriptively, organizations had more negative classifications of their tweets (46.5% vs. 40.4%), while managers had more positive classifications of their tweets (59.6%). Therefore, the initial hypothesis that sentiments of emergency dispatches/EOC's tweets would be more positive than the sentiments of tweets communicated from on-site by the managers was refuted.The difference in sentiment average score between managers and organizations was statistically significant, as observed in an independent samples t-test, Through content analysis, four categories of hashtags used during the crisis were identified, referring to: 1) geographical locations – e.g., #WestGate, #WestGateMall, #Kenya, #Nairobi. These hashtags existed and were used on Twitter prior to the attack; 2) terror/violent attacks– e.g., #WestGateAttack, #WestGateSiege, #WestGateMallAttack and #WestGateShootout. Kenyan government officials, organizations and first responders used #WestGateAttack only; 3) social support, resilience and cohesiveness– e.g., #WeAreOne and #UnitedWeStand. The latter, though used by the public was absent from all official tweets; and, 4) organizations – e.g., #RedCross and #AlShabaab.During the first days of the attack, numerous complaints were posted on Twitter stating that there were too many hashtags for the event or alternatively posting a list of hashtags, such as “One incident, many hashtags” or “So many different hashtags for today's tragedy #Westgate #WestgateMall #WestagateAttack #WestgateShootOut #WestGateMallShootOut”. Different variations were used inconsistently and interchangeably by the public, the media, and NGOs. On September 24 the PSCU Kenya Digital tweeted that all of the social media managers had held a synchronization meeting- “This morning we held a WhatsApp meeting with all bloggers running the @InteriorKE @PoliceKE @kdfinfo @NDOCKenya our voice is #WithOneAccord”, following which all responders consistently used the #WithOneAccord hashtag.Moreover, while the fourth category of hashtags was present in many tweets, government officials preferred to use the “@” handle to mention the different organizations and individuals in their messages, as shown in Mentioning was frequently used by government officials and responding organizations as presented in During this crisis, Twitter seems to have been the dominant social media platform used by the public and responding organizations. Though limited in scope, Facebook too was used. For example, the Kenya Police started posting on Twitter on September 21, but posted for the first time on its Facebook wall only on September 23, following an announcement on Twitter that included their Facebook page address http://twopcharts.com/howlongontwitter). Eight of the accounts were created between May and September 2013, two of which (Kenya Police and Kenya Military) joined Twitter less than ten days prior to the attack.As an integral component of responding to the crisis, a number of governmental officials, organizations, first responders, and NGOs tweeted information concerning the event. The different organizations are presented in @InteriorKE) and its minister Joseph Ole Lenku (@JoeLenku) communicating from the scene near the Westgate mall. Furthermore, both the Kenya Police and the Ministry of Interior tweeted throughout the event that official communications would be posted through their respective managers' Twitter accounts.In most organizations, communications were transmitted simultaneously by both the manager of the organization and the organization itself see . FrequenThe abundance of Twitter accounts providing official information regarding the event made it difficult to synchronize and follow the flow of information. For example, on September 22, the Kenya Police stated “For systematic flow of information on the operation at West Gate please follow @kdfinfo @InteriorKE @PoliceKE @IGkimaiyo @joelenku”, which was retweeted 108 times. On September 23, the Ministry of Interior requested the Kenyan Military to follow them back on Twitter th, The PSCU Kenya Digital held a WhatsApp synchronization meeting with all of the social media managers in charge of the governmental Twitter and Facebook accounts On the previous night, starting at 10:10 PM all government organizations started using the #WithOneAccord hashtag, initiated by the PSCU Kenya Digital account. On the following morning, September 24@EMS_Kenya). Prior to the attack, their account had been active on Twitter.One first responder organization that participated in the response phase and which was referred to in other organizations' tweets was the Red Cross ambulance services  producing information concerning the event, 2) sharing and consolidating information, 3) expressing emotional and social support for individuals, communities or responders affected by the crisis 4) sharing concerns about privacy and security issues regarding social media, 5) requesting or offering to volunteer, and 6) criticizing the government and its security services.The Al-Shabaab organization twitted 258 times between September 21 and September 25. They claimed responsibility for the attack on the Westgate mall through Twitter. The Al-Shabbab used a number of accounts to try to avoid being caught by Twitter, but often had to change accounts as Twitter suspended them for breaching their terms of use. We followed three different accounts that were active on different dates “@hsmpress_” (21–22/9), “@HSM_PRESOFFICE2” (22–23/9) and “@HSM_PR” (24–25/9). The Al-Shabaab tweeted from the “@hsmpress_” account regarding their suspension on September 22 – “Alshabab has suffered another great casualty: Twitter account loss!”.Using their Twitter accounts, all government organizations and officials provided the public and other responding organizations with situational awareness updates. These updates included five classifications of information, including 1) risk communication and public warnings – e.g., Kenyan Police tweeted an hour after the attack: “The public should stay away from West Gate and its environs and we ask the media to refrain from any unnecessary comments.” The Kenya military tweeted: “Report any suspicious looking individuals or activities to the nearest security organizations of the Govt”. 2) Status updates about the armed response towards controlling the mall – e.g., the Kenya Police tweeted: “We have taken control of the ground floor and we urge you to be patient”, or the Kenya military, which communicated that: “KDF has dominated all floors of Westgate Mall building. Troops are now concentrating on clearing the building”. 3) Information hubs for other organizations and officials. The public contributed to the situational awareness by tweeting information regarding the developments as they experienced them from the surroundings of the Westgate mall. Some even shared tweets with photos taken from the scene.All responding organizations used Twitter to communicate and notify other organizations during the crisis. This was done by adding a mention to a tweet with a “cc” before it (as in emails), i.e., a tweet sent out by the Ministry of Interior: “Give way to ambulances making their way to #WestgateMall cc @NDOCKenya”. This tweet was sent out to the general public, but it also notified the Kenyan Disaster Operation Center by mentioning it, which retweeted it approximately two hours later. Almost an hour and a half after the attack began, the Minister of Interior arrived at the scene and tweeted: “I am at the scene and urge the public to be calm, give support to @IGkimaiyo and @PoliceKE and we will resolve this quickly. cc @InteriorKE”. It was retweeted by the Ministry of Interior less than a minute later.The government organizations and officials were reading tweets that mentioned their names. In some cases they retweeted the original message, as the Police Inspector General retweeted: “RT @CitizenTVNews: Inspector General of Police David Kimaiyo via his twitter account @IGkimaiyo says the Westgate mall is currently surroun…”. The hypothesis that emergency organizations and managers new to Twitter would use it as a basic, one-way, communications channel was refuted. No differences were found in the use patterns of Twitter between new versus more experienced users.We identified two ways emergency authorities requested assistance from the public during the crisis: 1) a direct call for information – e.g., the Inspector General of the police requested information regarding potential hostages “Kindly share information of the families who have been shopping and have not been traced. “ 2) Requesting the public to retweet their messages or share information on Facebook, e.g., the Kenya police tweeted “Forensic probe is underway. We urge the public to stay away for their own safety. Pls RT.”At the beginning of the crisis, the responding organizations and the public did not understand the nature of the event. Stemming from the initial tweets, the police perceived that robbers rather than terrorists were attacking the mall. The Ministry of Interior tweeted approximately 30 minutes after the attack: “We've managed to evacuate some people to safety as @PoliceKE pursues the thugs”. Fifteen minutes later, they tweeted: “The IGP @IGkimaiyo is heading to the scene of crime to assess the situation. He'll also give a statement.” Approximately three hours after the attack began, Reuters tweeted that there was a possibility that terrorists might be perpetrating the attack.Both the responding organizations and the public were wary about operational security. Many bystanders and journalists on site tweeted and broadcast their operations in real time. One bystander tweeted “why is this our media showing us the cops sneaking in??.” The Kenyan military tweeted “We urge all Kenyans to be cautious on what is being communicated on social media.”, and the PSCU Digital Kenya tweeted “We are appealing to Media to avoid showing photos of our @kdfinfo soldiers. Kindly, Only tweet what you are absolutely sure about #Westgate.” In one instance the Kenya Police asked a Twitter user to delete a message that contained pictures of military helicopters preparing to launch an attack on the mall On the third day of the crisis, the police published a direct threat of prosecution to those sharing ‘repugnant’ information on Twitter - “Those who have shared content that is repugnant to our sense of nationhood be warned that you will be found and charged.”During the first day of the attack, two unique tweets were posted, claiming to show pictures of the attackers. These messages were retweeted 106 times, 81 of them within 30 minutes. We found that these pictures were not of the attackers but rather of the Kenyan armed forces. The photos were removed after approximately two days.Tweets can include the coordinates of the location the tweet was sent from. While government organizations and officials' accounts did not include geo-location information, 0.9% of tweets collected from the public included geo-location information. Out of all the accounts listed in The location of the managers was deduced based on the tweets' text or geo-location information. Although they did not include their own location they did include location based information in the text of the messages, whether with clearly identifiable information or with a location reference. One example of a tweet with a clear location can be found in a tweet by the Kenyan Disaster Operation Center 20 minutes after the attack had begun “Mwanzi Road between Peponi Rd and Ring Road Parklands is CLOSED TO ALL TRAFFIC.” The Ministry of Interior tweeted a message that included location based information by referring to an identifiable place “Roads adjacent to the #WestGateMall have been cordoned off, PLEASE use alternative route.”On the third day of the attack, Twitter contacted the Kenyan government so that they could verify their accounts used to publish information on the Westgate attack. PSCU Kenya Digital first tweeted about it “@twitter has shared its support by contacting @PSCU_Digital to verify Government accounts-we have started with those communicating #WestGate.” In the following three hours, most accounts of the Kenyan government organizations and officials, including the Kenya Red Cross, were verified.Prior to the verification process, the governmental organizations had to inform the public as to which accounts were sharing verified information, as tweeted by the Kenya Police on the second day “We urge all Kenyans to wait for official information from @InteriorKE @IGkimaiyo @joelenku @StateHouseKenya.”Our findings show that Twitter served as an integral tool for emergency management in Kenya; all emergency related organizations and officials had accounts and actively communicated through them during the crisis, both with the public and among themselves. The use of Twitter in Kenya is not restricted to emergency situations alone but rather characterizes routine activities throughout the year. Merchant et al. The response to the attack was managed and coordinated by the Kenyan Police, supported by other first responders including the Red Cross ambulance service, EMS, and the fire brigade. The Kenyan military arrived on the first day to support and enhance the response, and the Kenyan National Disaster Operations Center oversaw the entire operation.As presented in previous emergencies by different governments worldwide, the Kenyan government and ministers widely adopted the use of the social media during the terror attack and leveraged it in order to better understand the situation in real time, communicate with the public and improve their safety Service providers, and especially public officials, emergency responders and spokespersons In this study, we found 15 different Twitter accounts disseminating and communicating messages, which caused information overload and synchronization problems. Bharosa, et al. Official responders and organizations were not prepared to manage Twitter communications 24/7 during the crisis, and had long breaks during the night, sometimes for up to 10 hours. It was also apparent that some organizations did not know, or were unaware of the Twitter accounts of other responding organizations, creating gaps between the ministry and the military. In one case we found that the Ministry of Interior was unaware of the Kenyan Military's Twitter account, although it was active from the first day of the crisis. Most probably, this resulted from the Military tweeting messages without any hashtag or mentions, causing them to be visible only to their direct followers.The rapid rise in information available on Twitter within minutes after the initiation of the attack may be explained by two main factors: Twitter is highly used in Kenya and thus serves as a popular platform for sharing information Throughout the crisis civilians, reporters, and responding organizations, as well as the terrorists, shared information on Twitter about the status of the response. From the tweets it was clear that many media personnel were present in the vicinity of the mall, as were numerous bystanders. During emergencies it is common to access situational awareness updates from the responding organizations, the media and the general public on Twitter From this crisis we can learn that the ‘traditional’ two-way communications channel view of Twitter was used with a more extensive approach, as a cross-hierarchy, inter- and intra-organizational tool for communications and coordination of the response. All responding entities and their managers, whether ministers, managers or commanders, used it to communicate with the public, with their own organization and with other responding organizations. This indicates that the organizations have the support of their management to implement and use Twitter, but nevertheless, a standard operating procedure (SOP) is crucially needed for effective implementation in emergencies. The SOP should delineate ways to synchronize communication between official first responders and emergency organizations, such as the WhatsApp synchronization meeting that was conducted during the Kenyan terror attack. This online meeting included all bloggers managing the social media accounts of the government organizations, emphasizing the fact that the social media managers are not employees of the government. The SOP should also relate to the challenge of continuous communication during crises. The fact that during the night there was a pause in Twitter updates to the public, sometimes for up to ten hours, suggests that only one person operated the social media account, lacking replacements or organized shifts. Kenya's Police Twitter operator reported this in one of his tweets referring the public to other official accounts for updates – “After almost 36 hours without sleep, I'm off duty at Visa Oshwal Center. Please follow @kpsya @interiorke @policeke @kdfinfo.via @kangai” Publishing situational awareness updates through social media during emergencies is a common practice for numerous participants During the first day of the attack, many civilians published photos of what they thought were the attackers. Some of these photos were retweeted by hundreds, not knowing that they were incorrect. The same photos were published by news agencies, such as the New York Times, describing them as “plainclothes officers” The Kenyan officials and organizations tried to direct the public to their accounts for access to verified and reliable information, but this was not necessarily implemented. This reflects two main challenges of using social media in emergencies: Anyone can open an account on social media sites and publish information Social media offer an accessible, widely available means for bi-directional flow of information between the public and the authorities in charge of public safety. Their use is growing across geographies, cultures, languages and organizations. This manuscript portrays the use of social media by the various participants in an unexpected event, and focuses mainly on use by emergency organizations. Emergency management almost always entails the response of multiple organizations and NGOs; in this crisis, all of these used and leveraged social media networks, not solely for communicating with the public, but also among themselves, frequently using two different Twitter accounts (for managers and for organizations), and for both public and private communications. Twitter offers emergency entities a communications platform that is ubiquitous and does not have interoperability issues, like other communication systems. This event showed that such a scenario may cause synchronization and coordination problems affecting response, and may waste valuable resources. Lessons learned from this event should contribute to the development of an SOP that will allow multiple responders to monitor, synchronize and integrate their social media feeds during emergencies. This will lead to better utilization and optimization of social media resources during crises, providing clear guidelines for communications and a hierarchy for dispersing information to the public and responding organizations during emergency situations. We recommend that future research analyze the differences between routine and emergency utilization of the social media by emergency organizations.Limitations of the present study include the current state of sentiment analysis of crisis communication. Unfortunately, there is still a lack of studies analyzing emotional valence of micro-blog information during emergencies TwitterMate or similar instruments can become a tool that will help to manage and coordinate emergency events. Governments as well as emergency organizations should consider the availability of social media tools when they define their policies, and outline information they wish to share, and through what channels. Regulating the information provided by the public is yet another challenge that can possibly be better achieved through education rather than through regulation.


There is an error in the Funding Disclosure statement. The correct Funding Disclosure statement should read: "This work was supported by the National Natural Science Foundation of China (Grant No. 31401390) and the China Agriculture Research System (Grant No. CARS-02-01). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."


The occurrence of cancer during pregnancy merges two complex, poorly understood metabolic and hormonal conditions. This association can exacerbate the conditions of both the mother and the foetus. The branched-chain amino acid leucine enhances cellular activity, particularly by increasing protein synthesis. This study aimed to analyse the modulatory effect of a leucine-rich diet on direct and indirect tumour-induced placental damage. This was accomplished by evaluating the expression of genes involved in protein synthesis and degradation and assessing anti-oxidant enzyme activity in placental tissues collected from pregnant, tumour-bearing rats.th day of pregnancy, tumour growth was observed. Dams fed a normoprotein diet showed the greatest tumour growth. Injection with ascitic fluid mimicked the effects of tumour growth. Decreased placental protein synthesis and increased protein degradation were observed in both the tumour-bearing and the ascitic fluid-injected groups that were fed a normoprotein diet. These effects resulted in low placental DNA and protein content and high lipid peroxidation . Decreased placental protein synthesis-related gene expression was observed in the tumour group concomitant with increased expression of genes encoding protein degradation-associated proteins and proteolytic subunits.Pregnant rats were either implanted with Walker 256 tumour cells or injected with ascitic fluid (to study the indirect effects of tumour growth) and then fed a leucine-rich diet. Animals in a control group underwent the same procedures but were fed a normal diet. On the 20Consumption of a leucine-rich diet counteracted the effects produced by tumour growth and injection with ascitic fluid. The diet enhanced cell signalling, ameliorated deficiencies in DNA and protein content, and balanced protein synthesis and degradation processes in the placenta. The improvements in cell signalling included changes in the mTOR/eIF pathway. In conclusion, consumption of a leucine-rich diet improved placental metabolism and cell signalling in tumour-bearing rats, and these changes reduced the deleterious effects caused by tumour growth. Cancer is the second leading cause of disease-mediated death. The most common types of cancer in women are breast and cervical cancers , 2. NeopMoreover, the prevalence of cancer during pregnancy is not rare, and the coexistence of these two complex metabolic and hormonal conditions can negatively impact the mother and foetus , 4. To dThe endocrine changes associated with cancer evolution during pregnancy have not been well studied because of the many physiological adaptations that concomitantly occur in the maternal organism. These adaptations are primarily driven by the maternal/foetal unit known as the placenta , 10. Thuad libitum and were maintained under controlled light and temperature conditions . To obtain pregnant rats, inbred mating was performed using 4 females per 1 male. Mating was allowed to occur for 12 hours, and pregnancy was detected by assessing vaginal smears for the existence of sperm. The pregnant rats were subsequently distributed into 6 groups . Three groups were fed a control diet: a control group (C), Walker tumour-bearing group (W), and ascitic fluid-injection group (A). The remaining three groups were fed a leucine-rich diet: a leucine-fed control group (L), tumour-bearing group (LW), and ascitic fluid-inoculation group (LA). Each animal in the two tumour-bearing groups (W and LW) was subcutaneously injected with approximately 1 × 106viable Walker 256 tumour cells on the second day of pregnancy. Each animal in the ascitic fluid injection groups (A and LA groups) was injected with ascitic fluid from the 9th to the 20th day of pregnancy  phenylalanine/mL, Amersham) was added and incubated for an additional 2 h. Next, the placentas were removed from the medium, dried, weighed, homogenised in 30 % TCA (1:3 w/v), and centrifuged at 10,000 x g for 15 min at 4 °C. The pellet was washed twice with 10 % TCA to remove radioactivity from the supernatant and then suspended in 1 M NaOH and incubated at 40 °C for 30 min. Aliquots were analysed for protein content [3H]-phenylalanine per microgram of placental protein per hour [Fresh placental tissues were removed and individually placed into tubes for perfusion with Krebs-Henseleit buffer (KHB)  . Followiper hour .Another sample of fresh placenta was used to analyse protein degradation, which was expressed as nmol of tyrosine released per microgram of protein per hour. The placenta sample was pre-incubated for 30 min at 37 °C with RPMI-1640 medium without phenol. Following this, the sample was incubated with KHB buffer supplemented with 130 μg/mL cycloheximide (a protein synthesis inhibitor) for 2 h at 37 °C and then gassed with 5 % CO2 + 95 % O2. Next, an aliquot of the incubation medium was assessed for tyrosine content, which was measured fluorometrically using 1-nitroso-2-naphthol and nitric acid (20 %) according to a method published by Waalkes & Udenfriend [Placental tissue was homogenised in homogenisation buffer  and incubated for 1 h at 65 °C. Subsequently, 8 M potassium acetate was added, followed by incubation and centrifugation under refrigeration. DNA was extracted from the upper phase using phenol:chloroform:isoamyl alcohol (25:24:1) and then precipitated with 100 % ethanol overnight at −80 °C. DNA content was quantified by measuring the 260/280 nm absorbance ratio using a spectrophotometer. The coefficient of variation (CV) ranged from 6.5 % to for the control, tumour and ascitic fluid samples.Aliquots of placental homogenate supernatant were analysed for alkaline phosphatase (AP) activity using 37 mM 4-nitrophenyl disodium phosphate . AP activity was expressed in nmol/min/mg protein . For thi-ΔΔCt). We analysed the expression levels of the protein degradation-associated and protein synthesis-associated genes as described in Table Samples of placental tissue 10 mg) were lysed in 1 mL TRI reagent solution , and total RNA was extracted as described by the manufacturer. The RNA concentration was determined based on the absorbance at 260 nm. cDNA was synthesised from 3 μg of placental RNA in a volume of 20 μL using MultiScribe™ Reverse Transcriptase and random hexamer primers . To amplify cDNA using real-time PCR, reactions were performed in a total volume of 20 μL containing 5 mM each primer, 4 μL cDNA (20x diluted), 4 μL milli-Q H2O and 10 μL Fast SYBR Green 2X Master Mix . The placenta samples were denatured at 95 °C for 10 min, followed by 40 PCR cycles at 95 °C/60 °C. PCR amplification was performed in triplicate. Quantitative PCR assays were performed using a StepOne Real Time System . The purity of the amplified PCR products was verified based on melting curves. Target gene expression was analysed using 7500 Fast Real-Time PCR software, version 2.0.5 , and the values are expressed relative to control levels  followed by centrifugation at 10,000 × g for 15 min at 4 °C. Placental proteins (40 μg) were resolved by 12 % SDS-PAGE at 90 V for 1 h, followed by transfer onto 0.45-μm nitrocellulose membranes at 300 mA for 2 h. The membranes were blocked with 5 % non-fat dry milk in Tris-buffered saline (pH 7.5) for 1 h at room temperature. The membranes were incubated overnight at 4 °C with primary antibodies against the 20S, 19S and 11S proteasome subunits  as well as MuRF-1 (#SC-32920) and MAFbx . Immunoreactivity was detected by sequentially incubating the membranes with specific secondary antibodies  for 1 h at room temperature and visualised using a chemiluminescence detection system. The blots were scanned using a gel image capture system to quantify differences via densitometry . The levels of all detected proteins were reported relative to the level of 34-kDa GAPDH.Placenta samples were homogenised in protein extraction buffer . Inter-group comparisons were made using the post hoc Bonferroni multiple-comparison test. The results were considered significant when P < 0.05 .P < 0.0045). However, injections with ascitic fluid had no effect on body weight. Additionally, regardless of tumour presence, the leucine-treated group (WL) exhibited better body weight recovery compared to the C and L groups . As in our previous experiments, a significant tumour effect leading to a reduction in the number of foetuses was observed . Additionally, tumour-bearing, pregnant animals exhibited increased foetal resorption and reduced foetal weight (Table P < 0.0047). Similarly, injections with ascitic fluid resulted in decreased foetal number and weight and increased foetal resorption . Tumour-bearing rats fed a leucine-rich diet (WL) exhibited a non-significantly decreased number of foetuses and increased foetus resorption relative to the control group (C); however, average foetal weight was significantly reduced compared to the C and L groups. Consumption of a leucine-rich diet minimised the effects caused by ascitic fluid injection: foetal resorption rate were maintained. The interaction between ascitic fluid and diet accounted for 20 % of the variation, which was significant (P < 0.0321); however, the diet could not correct for low foetal weight. The relationship between foetal weight and placental weight is indicative of the efficiency of placental transport . In contrast, in the LW group, there was a positive correlation between placental and foetal weight. This relationship indicated that placental weight influenced foetal weight in this group .Tumour growth produced harmful effects on body weight evolution in the W group: a significant variation of 31 % was observed . In addition to the above tumour effects, placental protein synthesis decreased in both the tumour-bearing (W) and ascitic fluid-injected groups (A) . Consumption of a leucine-rich diet significantly increased placental phenylalanine incorporation in the L group (P = 0.0452) but either had no effect or reduced phenylalanine incorporation in the LW and LA groups . This result indicates reduced balance of total protein net in the tumour-bearing and ascitic fluid-injected groups  Fig. . The tumups Fig. . The tum378 Fig. . Consumpups Fig. .Fig. 2PlP = 0.003). Placental glutathione-S-transferase (GST) activity was only reduced in W group tumour-bearing dams . Consumption of a leucine-rich diet significantly increased placental GST activity in the LW and LA groups versus the L group . The reduced glutathione levels were constant among the groups . Additionally, the interaction between tumour/ascitic fluid and diet produced an extremely significant effect . The MDA/GST ratio is an indicator of damage induced by poor cellular responses to external effects. The W group exhibited a significantly higher MDA/GST ratio relative to the other groups; the tumour effects in this group accounted for the significant difference (P = 0.0065). Such differences were not observed in the leucine-treated tumour-bearing or ascitic fluid-injected groups, in this case the interaction between tumour/ascitic fluid and diet had a significant effect .Overall cellular activity can be correlated with AP activity , 20. Tumups Fig. . OxidatiP < 0.05). Ascitic fluid injection (A group) mimicked the indirect effects of tumour growth, including reductions in the expression of eIF4E and 4EBP1 . The leucine-supplemented groups (LW and LA) exhibited similar profiles that accounted for tumour/ascitic effects   Fig.  and g.FiP = 0.027, P = 0.0147 and P = 0.0216). Similarly to the tumour/ascitic effect, we also observed increased expression of the MuRF-1 and ubiquitin genes in the LW group . We also observed increased 11S protein expression in the L and LW groups, which were fed a leucine-rich diet; the effect of the diet was significant . No differences were observed among the groups with respect to 19S and eIF2a (Fig. Protein degradation also increases during tumour growth. Several key proteins were highly expressed in the placental tissues of the animals in the W group. These included MuRF-1, ubiquitin, ubiquitin-proteasome pathway enzymes, and calpain, which is related to the calcium-dependent proteolysis pathway. Tumour growth was responsible for highly significant effects (Fig. oup Fig.  and e anF2a Fig.  and g.FiTumour development, especially development of Walker 256 tumours, which serve as an experimental model of cachexia , 22, proFoetal growth is primarily determined by nutrient availability, which is related to the capacity of the placenta to transport nutrients , 30. FoeSeveral reports have suggested that placental oxidative stress is involved in the etiopathogenesis of pregnancy-related diseases, such as preeclampsia, as well as in foetal growth restriction , 35–37. Placental function is regulated by numerous factors of foetal, maternal, and placental origin , 39–43. Placental trophoblast cells must integrate numerous and possibly divergent maternal and foetal stimuli and modify cellular functioning according to the host-environment interaction, as we have previously reported , 28. PreThe inoculation of ascitic fluid and the effects of humoural factors mimicked the results of PIF and WF in tumour-bearing rats . We prevOur study highlights the important role of a leucine-rich diet in modulating placental cell activity and maintaining antioxidative response during tumour growth. As many other studies have proposed, leucine, one of the three branched-chain amino acids, can act as a cellular signal to increase protein synthesis downstream of mTOR, p70S6K1 and eIF4G in skeletal muscle –47. In aAs in previous studies in our laboratory, we found that tumour growth promotes intense mobilisation of muscle proteins associated with diminished protein synthesis –27. TumoIn the current study, we demonstrated that dietary leucine supplementation enhances placental cell signalling, ameliorates deleterious alterations in DNA and protein content, and increases placental cell number. Leucine supplementation also improved the balance between protein synthesis and degradation in the placenta. This was reflected in enhanced cell signalling, including signalling via the mTOR/eIF pathway. Additional experiments are being conducted to better understand the effects produced by ascitic fluid injection and to determine the mechanisms by which leucine improves placental metabolism and counteracts the effects of tumour growth.


During cold weather months in much of the country, the temperatures in which prehospital care is delivered creates the potential for inadvertently cool intravenous fluids to be administered to patients during their transport and care by emergency medical services (EMS). There is some potential for patient harm from unintentional infusion of cool intravenous fluids. Prehospital providers in these cold weather environments are likely using fluids that are well below room temperature when prehospital intravenous fluid (IVF) warming techniques are not being employed. It was hypothesized that cold ambient temperatures during winter months in the study location would lead to the inadvertent infusion of cold intravenous fluids during prehospital patient care.Trained student research assistants obtained three sequential temperature measurements using an infrared thermometer in a convenience sample of intravenous fluid bags connected to patients arriving via EMS during two consecutive winter seasons (2011 to 2013) at our receiving hospital in Syracuse, New York. Intravenous fluids contained in anything other than a standard polyvinyl chloride bag were not measured and were not included in the study. Outdoor temperature was collected by referencing National Weather Service online data at the time of arrival. Official transport times from the scene to the emergency department (ED) and other demographic data was collected from the EMS provider or their patient care record at the time of EMS interaction.b = 0.69, t(21) = 4.3, p < 0.001. Transport time did not predict the measured intravenous fluid temperatures, b = 0.12, t(20) = 0.55, p < 0.6.Twenty-three intravenous fluid bag temperatures were collected and analyzed. Outdoor temperature was significantly related to the temperature of the intravenous fluid being administered, Use of unwarmed intravenous fluid in the prehospital environment during times of cold ambient temperatures can lead to the infusion of cool intravenous fluid and may result in harm to patients. Short transport times do not limit this risk. Emergency departments should not rely on EMS agencies’ use of intravenous fluid warming techniques and should consider replacing EMS intravenous fluids upon ED arrival to ensure patient safety. Many urban and rural emergency medical service (EMS) systems experience freezing temperatures on a regular basis. The United States National Oceanic and Atmospheric Administration (NOAA) National Climatic Data Center reports with 90% probability that our receiving hospital located in Syracuse, New York (NY) experiences a temperature at or below freezing 215 days per year and more generalizable, the entire Northeast, most of the Midwest, and the Great Plains north of Nebraska all experience similar temperature patterns with regard to number of days below freezing [Gentilello has equated the effect of unwarmed intravenous fluids (IVFs) on body temperature to that of a patient who must use 16 kcal of energy to warm a liter of room temperature 21°C) crystalloid to 37°C. If a 70-kg patient is unable to generate that heat, a decrease in body temperature of 0.3°C occurs which is enough to initiate shivering 1°C cryst.Intravenous fluid warmers are abundant in the commercial market, and ‘homemade’ methods of warming IVFs abound ,6. StillWe prospectively measured the temperature of intravenous fluids being administered to patients arriving to our emergency department by EMS. Trained student research assistants obtained a convenience sample of observations during two consecutive winter seasons (2011 to 2013) at our receiving hospital in Syracuse, New York (NY).Temperatures of the intravenous fluid bags were measured as soon as possible after entering the emergency department. Three sequential temperature measurements were taken by aiming an infrared laser thermometer  approximately 2 to 3 cm away from the fluid’s bag. During data organization, the average of these three measurements was calculated. Intravenous fluids contained in anything other than a standard polyvinyl chloride bag were not measured and were not included in the study.http://www.weather.gov) for our zip code (13210) at the time of arrival using a hospital computer. Official transport times from the scene to the emergency department and other demographic data were collected from the EMS provider or their patient care record at the time of EMS interaction.Outdoor temperature was collected by referencing National Weather Service online data . One patient’s transport time was not recorded but temperature data was used in analysis. Table The temperature data from 23 patients presenting by EMS were obtained and analyzed  = 4.3, p < 0.001. The outdoor temperature measured also explained a significant proportion of variance in the measured temperature of the IVF, R2 = 0.47, F = 18.64, p < 0.001. Conversely, the transport time did not predict the measured IVF temperatures, b = 0.12, t(20) = .55, p < 0.6. Figure The outdoor temperature measured significantly predicted the temperature of the IVF being administered,  0.69, t2 = 4.3, pAlthough prospective trials conflict on the effect of hypothermia on mortality, some effect on morbidity have been shown ,4. HypotOur data also show that transport time cannot be used to predict the temperature of IVF despite an inclination that short transport times might be protective of the fluid bag temperatures. We believe this data uncovers a possible misperception by transporting EMS agencies: specifically, that warming of their IVF stock is unnecessary within a small coverage area with relatively short transport times. On the contrary, we believe the data suggest that all EMS agencies who operate in cold climates would likely benefit from a methodology to keep intravenous fluids warm while they are stocked on an ambulance. It is likely that the temperature of the IVF is more a function of storage time than of administration time. Receiving emergency departments should be aware of this phenomenon.en route to a call [Lyng et al. demonstrated that liter bags of IVF could be warmed by the dashboard heater of an ambulance o a call . While to a call . EmergenWe believe that the strength of prediction between outdoor temperature and IVF temperature may have been stronger with a larger dataset. In our sample, one fluid bag was measured at 31.3°C which was considerably warmer than ambient temperature outside at that time (14°C). It is likely that this patient’s fluid had been warmed before or during transport, yet our study was not designed to capture these details and confounders. Future investigations should focus on EMS compliance with best practices of delivering warmed fluids to patients in cold winter environments in order to inform the practice of emergency nurses receiving these patients.This study is limited by the nature of convenience sampling. Our research assistants’ schedule was managed independent of the needs of this particular study; however, it is unlikely that more structured sampling would affect our results. Although other methods of measuring the IVF more directly are possible, we feel that our measurements were likely accurate. Indeed, infrared thermometry as employed in this investigation has been shown to be accurate for measuring temperatures of intravenous crystalloid fluid .In the interest of patient safety, it may be appropriate for emergency departments to consider polices that require the replacement of EMS intravenous fluids with hospital intravenous fluids that have been stored at temperatures closer to normal body temperature. In addition, these policies should take into consideration the amount of fluid administered by EMS as additional patient warming may be clinically indicated.The lack of EMS agencies’ use of intravenous fluid warming devices during winter months could contribute to mild hypothermia and possibly portend a deleterious effect on health. Even short transit times do not seem to protect against this risk. Use of IVF warming devices by EMS may not be routine.


Lysyl oxidase (LOX) and LOX-like (LOXL) enzymes are key players in extracellular matrix deposition and maturation. LOX promote tumour progression and metastasis, but it may also have tumour-inhibitory effects. Here we show that orthotopic implantation of rat prostate AT-1 tumour cells increased LOX and LOXLs mRNA expressions in the tumour and in the surrounding non-malignant prostate tissue. Inhibition of LOX enzymes, using Beta-aminopropionitrile (BAPN), initiated before implantation of AT-1 cells, reduced tumour growth. Conversely, treatment that was started after the tumours were established resulted in unaffected or increased tumour growth. Moreover, treatment with BAPN did not suppress the formation of spontaneous lymph node metastases, or lung tumour burden, when tumour cells were injected intravenously. A temporal decrease in collagen fibre content, which is a target for LOX, was observed in tumours and in the tumour-adjacent prostate tissue. This may explain why early BAPN treatment is more effective in inhibiting tumour growth compared to treatment initiated later. Our data suggest that the enzymatic function of the LOX family is context-dependent, with both tumour-suppressing and tumour-promoting properties in prostate cancer. Further investigations are needed to understand the circumstances under which LOX inhibition may be used as a therapeutic target for cancer patients. Loxl4 expression in bladder cancer using miRNA-193a-3p retards tumour growth172192122LOX as a tumour suppressor gene2425The Lysyl oxidase (LOX) family has five members, LOX and Lysyl oxidase-like (LOXL) 1–4, which are all secreted copper-dependent amine oxidases with the primary function to covalently cross-link collagen and elastin in the extracellular matrix (ECM), though intracellular functions of LOX have also been reported1256712LOX mRNA is associated with high-grade prostate tumours and tumour recurrenceLOX mRNA expression has been found to be lower in metastatic prostate tumours than in primary prostate tumours31LOXL2 genein situ hybridization and transcriptomics we showed that LOX was synthesized both in tumour epithelial cells and in epithelial cells in the non-malignant normal parts of the tumour-bearing organThe role of the LOX family in prostate tumour progression is largely unknown. High expression of The aim of this study was to investigate the expression of LOX and LOXL enzymes in orthotopic rat prostate tumours and in the tumour-adjacent normal prostate tissue, and how Beta-aminopropionitrile (BAPN) treatment . This shows that BAPN treatment did not affect metastatic capacity in the model tested. Only one animal in the control group and two animals in the BAPN-treated group had microscopic metastatic lesions in their lungs at this time point.To determine whether BAPN treatment had any effect on metastatic ability, the animals with orthotopic tumours were examined for the presence of lymph node and lung metastases. No metastases were detected in the lymph nodes or lungs 10 days after tumour cell implantation. Although AT-1 is not a highly metastatic tumour, we detected spontaneous lymph node metastases in both BAPN treated animals (5/7) and control animals (5/6) on day 16 after intra-prostatic implantation of tumour cells . In addiTo simulate metastatic colonization in the lungs, AT-1 tumour cells were injected in the tail vein and BAPN treatment was started on day 6 and continued on a daily basis until day 16. There was no significant difference between lung tumour burden in BAPN-treated animals and that in the controls , and onlin vitro under both hypoxic and normoxic conditions. BAPN treatment did not affect tumour cell viability under normoxic or hypoxic conditions compared to untreated controls , their inhibitors (Timp) and other proteases. Expression-levels changed more that 2-fold compared to tumour-free (vehicle injected) normal prostate tissue controls and with a p-value <0.05 were considered. In tumours, Collagen 1a1 (5.0 fold), Collagen 3a1 (5.0 fold), Collagen 4a1 (2.4 fold), Collagen 4a3 (2.1 fold), Collagen 5a1 (4.6 fold), Collagen 5a2 (11 fold), Collagen 18a1 (7 fold), Mmp3 (21 fold) Mmp14 (4.5 fold), Timp1 (2.5 fold), Cathepsin L (2.4 fold), Cathepsin S (2.3 fold), uPa (2-fold), Prolyl 4-hydroxylase alpha 1 (P4Ha1)(3.6 fold) and P4hb (−1.7 fold) were altered compared to controlsCollagen 1a1 (4 fold), Collagen 1a2 (2.4 fold), Collagen 3a1 (5 fold), Collagen 4a1 (3.8 fold), Collagen 5a1 (3 fold), Collagen 5a2 (3.8 fold), Collagen 15a1 (2.5 fold), Collagen 28a1 (2.2 fold), Mmp3 (2.6 fold), Mmp14 (2.7 fold), Timp1 (3.3 fold), Elastin (3.4 fold), Cathepsin L (1.8 fold), Cathepsin S (2.4 fold), uPa (2 fold), P4ha1 (1.8 fold) showed changed expression compared to control prostate tissueHypoxia has been suggested to stimulate collagen degradation in tumourset al. showed that metastatic formation was inhibited when treating mice with pancreatic cancer with a LOX-blocking antibody, while no survival effect was shown with the antibody alone, indicating that the primary tumour was unaffected by the treatmentMmps and other proteases, were increased in tumour and in tumour-adjacent non-malignant prostate tissue in ratsLOX has been shown to be an important regulator of the ECM, for pre-metastatic niche formation, and for metastatic establishment and growth of metastases5689171822233637383940In patients we have noted that high LOX in tumour epithelial cells was associated with high Gleason score but not with survival, whereas high LOX in non-malignant prostate epithelial cells was associated with a poor outcomeIn this study treatment of established tumours with BAPN increased tumour growth. The reason to this is unknown but one possible explanation could be that increased LOX activity in the tumour-bearing organ, in contrast to the early tumour-promoting role in the tumour microenvironment, could be part of an insufficient defence trying to seal off the tumour. Inhibition of this defence in situations where the tumour-promoting role of LOX within the tumour is of limited importance may therefore actually speed tumour growth. Alternatively, in tumours with low collagen content, other tumour suppressing functions of LOX may be dominating. Further studies are needed to study these possibilities.Loxl3—were up-regulated both in the tumour tissue and in the normal prostate tissue adjacent to the tumour. Thus, remodelling of the ECM by all of the LOX enzymes, by cross-linking collagen, might prepare the tumour-adjacent normal prostate tissue for tumour expansion and invasion or as suggested also be part of a defence mechanism induced to repair and strengthen the normal tissue. Further studies are needed to determine whether the different LOX enzymes have different functions in tumours or if they are able to compensate for each other. Whether or not there is a difference in the functions of these enzymes in tumours relative to their functions in the surrounding tissues is also unknown, and should be investigated further. As all LOX-family enzymes, except LOXL3, are upregulated under conditions of hypoxia6434445in vitro increased the mRNA expression of Lox, Loxl1, and Loxl2, suggesting that hypoxia also increased their expression in vivo. Hypoxia is also likely to upregulate the expression of LOX-family enzymes in the normal prostate tissue, especially Lox and Loxl2, as hypoxia increased their mRNA expressions in rat prostate fibroblasts in vitro.The LOXL proteins have not been thoroughly studied in tumourigenesis. If expressed, they are likely to have functions similar to those of LOX, both as tumour suppressors and tumour promoters135152Loxl2 in a breast cancer model stimulated tumour invasiveness4in vitro, suggesting that LOX does not affect the AT-1 tumour cells directly. The effects of BAPN treatment in vivo are therefore probably due to inhibition of LOX activities in the tumour stroma and in the surrounding prostate tissue.Intracellular localization of LOX has been observed in several cell lineages, suggesting that LOX may also have enzymatic activity inside cells12In summary, functional studies inhibiting LOX enzymes in an orthotopic prostate cancer model resulted in opposite effects, with reduced tumour growth if the treatment was initiated before to tumour cell implantation, and increased tumour growth if the treatment was initiated when tumours were already established. Anti-LOX treatment did not affect the growth of metastases in lymph nodes or lungs. The paradoxical roles of the LOX family therefore need to be studied further, to enhance the tumour-suppressive effects while inhibiting the tumour-promoting ones.3 in 10 μl RPMI 1640) or vehicle (10 μl RPMI 1640) were carefully injected into one lobe of the ventral prostate of adult male Copenhagen rats  as previously describedDunning R-3327 AT-1 rat prostate tumour cells  were grown in culture as previously described3) were injected into rat prostate and the rats were treated with intraperitoneal injections of the LOX inhibitor BAPN  or PBS (matching controls). Three different experiments were performed for orthotopic tumours: (1) treatment started one day before to tumour cell injection and continued on a daily basis until sacrifice on day 10 ; (2) treatment started on day 6 continued on a daily basis until sacrificed on day 10 ; and (3) treatment started on day 6 and continued on a daily basis until sacrifice on day 16 . The prostate lobes containing the tumours were removed, weighed, formalin-fixed and paraffin-embedded. The lymph nodes and lungs were removed and formalin-fixed to study spontaneous formation of metastases. Furthermore, AT-1 cells  were injected in the tail vein to simulate metastatic colonization in the lungs, and treatment started on day 6 and continued on a daily basis until sacrifice on day 16 . For small orthotopic tumours growing within the ventral prostate tissue (treatment from day -1 to 10 and day 6–10), tumour area was assessed in haematoxylin- and eosin-stained prostate tissue sections using the Pannoramic Viewer software version 1.15 . The section with the largest tumour area in each animal was chosen to represent tumour size. For large orthotopic tumours that had outsized the normal prostate tissue and predominantly contained tumour tissue (treatment from day 6–16), tumor weight represent tumor size. For lymph node metastases, total tumour area in all lymph node tissue in each animal was analyzed on representative sections and represented the lymph node tumour size. For tumours in the lungs, tumour size is given as the total area of all tumours in a representative section of each lung. Animal experiments were carried out in accordance with protocols approved by the Umeå Ethical Committee for animal studies (permit number A110-12).For LOX inhibition studies, AT-1 cells , corresponding adjacent non-malignant prostate tissue , tumour-free vehicle-injected control prostate tissue (n = 7), and AT-1 cells in vitro (n = 2 different cell culture flasks), using the TRIzol method according to the manufacturer’s directions (Invitrogen). The RNA was DNase-treated  to remove contaminating DNA, and 0.8 μg was used for synthesis of cDNA using Superscript II according to the manufacturer’s instructions (Invitrogen). Real-time qRT-PCR was performed using the Applied Biosystems 7900HT Real-Time PCR System and Taqman Gene Expression Assay (Applied Biosystems). Quantification of mRNA levels was done in a 20-μl reaction volume with 20 ng cDNA per reaction, with commercially available primer and probes  (Applied Biosystems). Negative controls were run in parallel, and the relative values for each gene were normalized using beta-actin (Rn00667869_m1) as reference gene. We used Taqman Analysis Software SDS2.4 (Applied Biosystems).Frozen prostate tissues, containing both AT-1 tumour and surrounding non-malignant tissue, were sectioned and stained with haematoxylin and eosin to determine tumour location. Using the sections for guidance, frozen tumour tissue and surrounding non-malignant tissue were separated and dissected with a surgical blade. To avoid contamination with tumour tissue, the non-malignant tissue was taken with a spacing of 1 – 2 mm from the tumour. Total RNA was then extracted from AT-1 tumour tissue  and 10 (n = 7), and tumour-free controls (n = 5) were stained with Sirius red and examined by polarization microscopyRat prostate tissues were stained for LOX using the automatic staining system Ventana Benchmark Ultra . Briefly, 4-μm-thick paraffin sections were pretreated with CC1 for antigen retrieval and stained with primary polyclonal antibodies to LOX . Samples were visualized using the ultraView Universal DAB Detection Kit. Sections were analysed using Pannoramic Viewer software. To visualize collagen fibres, paraffin sections from AT-1 tumours at day 7  and cytokeratin-5 and -18 negative . AT-1 and primary rat fibroblasts were grown in 6-well plates at 37 °C for 24 h under hypoxia  or under normoxia . Six replicates were used when total RNA was prepared, and Lox and Loxl1-Loxl4 mRNA levels were examined as described above.Primary rat mesenchymal cells, referred to as fibroblasts, were isolated from the ventral prostate of tumour-free Copenhagen rats as previously described3 cells/well) were seeded on a 96-well plate in complete medium and incubated in 37 °C over night. The following day, the indicated concentrations of BAPN  or recombinant LOX protein (Origene)  were added to the cells (6 replicates/concentration/time-point) and the cells were incubated for 24–96 hours in normoxia or for 24–48 hours in hypoxia as described above. MTT labelling reagent and solubilisation solution was added at the different time-points and absorbance was measured on the following day at 550 nm with a reference wavelength of 650 nm according to the manufacturer’s protocol.Cell viability was determined with an MTT 3--2,5-diphenyltetrazolium bromide assay . Briefly, AT-1 cells .Values are presented as mean ± SEM and the Student t-test was used for comparison between groups. The level of statistical significance was defined as How to cite this article: Nilsson, M. et al. Inhibition of Lysyl Oxidase and Lysyl Oxidase-Like Enzymes Has Tumour-Promoting and Tumour-Suppressing Roles in Experimental Prostate Cancer. Sci. Rep. 6, 19608; doi: 10.1038/srep19608 (2016).


Comparison with the study of formation of chlorophenoxy radicals (CPRs) from chlorophenols (CPs) clearly shows that the thiophenoxyl-hydrogen abstraction from CTPs by H is more efficient than the phenoxyl-hydrogen abstraction from CPs by H, whereas the thiophenoxyl-hydrogen abstraction from CTPs by OH is less impactful than the phenoxyl-hydrogen abstraction from CPs by OH. Reactions of CTPs with H can occur more readily than that of CTPs with OH, which is opposite to the reactivity comparison of CPs with H and OH.The chlorothiophenoxy radicals (CTPRs) are key intermediate species in the formation of polychlorinated dibenzothiophenes/thianthrenes (PCDT/TAs). In this work, the formation of CTPRs from the complete series reactions of 19 chlorothiophenol (CTP) congeners with H and OH radicals were investigated theoretically by using the density functional theory (DFT) method. The profiles of the potential energy surface were constructed at the MPWB1K/6-311+G//MPWB1K/6-31+G level. The rate constants were evaluated by the canonical variational transition-state (CVT) theory with the small curvature tunneling (SCT) contribution at 600–1200 K. The present study indicates that the structural parameters, thermal data, and rate constants as well as the formation potential of CTPRs from CTPs are strongly dominated by the chlorine substitution at the  Therefore, they have similar geochemical behavior, toxicity, and physicochemical properties in the environment [Polychlorinated dibenzothiophenes (PCDTs) and polychlorinated thianthrenes (PCTAs) are analogues of dibenzofurans (PCDFs) and polychlorinated dibenzo-ironment ,5,6,7,8.ironment ,10,11,12PCDT/TAs were never intentionally synthesized for commercial purposes, but are formed as byproducts from the chemical processes that are similar to those resulting in the formation of PCDD/Fs. The major known sources of PCDT/TAs in environment are combustion, emissions from municipal and hazardous waste incinerators as well as industrial incinerators ,14. HighChlorophenols (CPs) are structurally similar to PCDD/Fs and the most direct precursors of PCDD/Fs ,20,21,223P), or Cl under high-temperature oxidative conditions. As yet, very little work has been done at the high temperatures relevant to these reactions.Similar to the formation of PCDD/Fs from CP precursors, the gas-phase formation of PCDT/TAs from CTP precursors was also proposed involving radical-radical coupling of two CTPRs and radical-molecule recombination of CTPR and CTP. The recent works have shown that radical-radical coupling are more competitive thermodynamically than radical-molecule recombination for the PCDT/TA formation ,31,32,33In recent research from this laboratory, we investigated the formation of chlorophenoxy radicals (CPRs) from the reactions of CPs with H and OH radicals ,35, basesyn and anti-conformers for 2-CTP, 3-CTP, 2,3-DCTP, 2,4-DCTP, 2,5-DCTP, 3,4-DCTP, 2,3,4-TCTP, 2,3,5-TCTP, 2,3,6-TCTP, 2,4,5-TCTP, 2,3,4,5-TeCTP and 2,3,4,6-TeCTP, respectively. The conformer with the sulfydryl-hydrogen facing the closest neighboring Cl is labeled as the syn-conformer and otherwise the anti-conformer , six dichlorothiophenols , six trichlorothiophenols , three tetrachlorothiophenols , and pentachlorothiophenols (PCTP). Due to the asymmetric chlorine substitution, there are onformer . For a gortho-substituted chlorine regardless of the number of chlorine substituents. There exists weak intramolecular hydrogen bonding in the ortho-substituted CTPs. The lengths of the intramolecular hydrogen bonds are from 2.391 to 2.490 Å. No such intramolecular hydrogen bonding forms in the anti-conformers except those with chlorine substitutions at both ortho-positions. The C–S bonds in CTPs are from 1.749 to 1.761 Å, which are longer than the C–S double bond and shorter than the C–S single bond. The C–S bonds lengths (1.749–1.754 Å) in the ortho-substituted CTPs are consistently shorter than those for all nonortho forms (1.756–1.761 Å). The structures of CTPRs along with the structure of thiophenoxy radical are shown in the The structures of CTPs along with the structure of thiophenol are presented in the S and NBOH), NBO charge of O and H of CPs (NBOO and NBOH) and HOMO-LOMO gap of CTPs and CPs at MPWB1K/6-31+G level. At a given CP and CTP, the NBOO of CP is more negative than the NBOS of CTP, and the NBOH of CP is more positive than that of CTP. This means the O atom in CP have stronger nucleophilicity than the S atom in CTP, i.e., the O–H bond strength in CP is stronger than the S–H bond in CTP. In addition, the HOMO-LOMO gap of CP is larger than that of CTP, which reconfirms that CP is more stable than CTP. et al. using both experimental investigation and ab initio molecular calculations at B3LYP/aug-cc-pVQZ, MP2(full)/aug-cc-pVTZ and MP2(full)/aug-cc-pVQZ levels [Comparison of C–S bond lengths presented in Z levels . The S–HZ levels . CompareZ levels . with H proceeds via a direct hydrogen abstraction mechanism. The structures of the transition states were located at the MPWB1K/6-31+G level and shown in ortho-substitution  are systematically shorter than those without ortho-substitution , respectively. Besides, all the ortho-substituted transition states have relative longer S–H bonds lengths (1.395–1.405 Å) compared to those without ortho-substitution (1.387–1.393 Å). ortho-substituted CTPs are consistently higher than those for other structural conformers. The chlorine substitution at the ortho-position can lower the barrier heights of thiophenoxyl-hydrogen abstraction from CTPs by H. Intramolecular hydrogen bonding appears to stabilize the CTPs and reduce the reactivity of S–H bonds in CTPs with the ortho-substitution. A similar result was also observed in our previous study of CPs with H [The formation of CTPRs from the reactions of CTPs D0 (S–H). The values of D0 (S–H) obtained at the MPWB1K/6-311+G//MPWB1K/6-31+G level are summarized in D0 (S–H) of 2-CTP is higher than those of 4-CTP. Similarly, D0 (S–H) of 2,3-DCTP, 2,4-DCTP, 2,5-DCTP and 2,6-DCTP are higher than that of 3,4-DCTP. D0 (S–H) of 2,3,4-TCTP, 2,3,5-TCTP, 2,3,6-TCTP, 2,4,5-TCTP and 2,4,6-TCTP are higher than that of 3,4,5-TCTP. The chlorine substitution at the ortho-position appears to increase the strength of the S–H bonds in CTPs. However, for a given number of chlorine substitutions, the S–H bond dissociation energies in CTPs with ortho-substitution are not consistently larger than those without ortho-substitution. For example, D0 (S–H) of 2-CTP is smaller than that of 3-CTP. D0 (S–H) of 2,3-DCTP, 2,4-DCTP and 2,6-DCTP are smaller than that of 3,5-DCTP. Chlorine in an aromatic ring is traditionally recognized as an electron-withdrawing group. The intramolecular hydrogen bonding in the ortho-substituted CTPs as well as the inductive effect of the electron-withdrawing chlorine and steric effect may ultimately be responsible for the relative strength of the S–H bonds in CTPs.In order to further investigate the relative strength of the S–H bonds in CTPs, we also calculated the S–H bond dissociation energies It is interesting to compare the thiophenoxyl-hydrogen abstraction from CTPs by H with the phenoxyl-hydrogen abstraction from CPs by H . For a gortho-substituted structures and nonortho-substituted structures. In the ortho-substituted intermediates, H(1) atom is at the trans-position of O with respect to the O–H(2) bond. In contrast, H(1) atom is at the cis-position of O in the intermediates without ortho-substitution. In addition, the ortho-substitution also has an effect on other structural parameters, such as the H(1)–O, H(2)–S and C–S bonds. For example, all the ortho-substituted intermediates have relatively shorter C–S bond distances (1.748–1.757 Å) compared to those without ortho-substitution (1.756–1.761Å). The relative energy, ∆EIM, of the intermediate with respect to the total energy of the corresponding CTP and OH is listed in For thiophenoxyl-hydrogen abstraction from CTPs by OH radical, prereactive intermediates are formed before the transition state. The structures of the prereactive intermediates are presented in trans-position of O with respect to the O–H(2) bond in the transition states with or without ortho-substitution. There are exit weak intramolecular hydrogen bondings in all the structures, which are governed by the chlorine substitution pattern. In the ortho-transition states, the intramolecular hydrogen bondings are between H(1) and ortho Cl atoms. In the transition states without ortho-substitution, the intramolecular hydrogen bondings are between O and H(3) atoms. The hydrogen bond can lower the energy of the transition state, i.e., lower the reaction potential barrier. Besides, the ortho-substitution also impacts other essential structural parameters of the transition states. Generally, the breaking S–H(2) bonds in the ortho-substituted transition states (1.437–1.456 Å) are longer than those without ortho-substitution (1.414–1.431 Å). The forming O–H(2) bonds in the transition states with ortho-substitution (1.382–1.419 Å) are shorter than those without ortho-substitution (1.420–1.464 Å).The structures of the transition states are depicted in ortho-substituted CTPs by OH radicals consistently are higher than those from CTPs without ortho-substitution. This reaffirms the conclusion above that the chlorine substitution at the ortho position increases the strength of the S–H bonds and decreases its reactivity.The potential barriers and reaction heats calculated at the MPWB1K/6-311+G//MPWB1K/6-31+G level are shown in i.e., higher the potential energy of CTPs with OH radicals. It is also necessary to compare the thiophenoxyl-hydrogen abstraction from CTPs by OH with the phenoxyl-hydrogen abstraction from CPs by OH of our previous study . For a gComparison of the values presented in 2 and phenol + OH → phenoxy + H2O are in good agreement with the available experimental values [Canonical variational transition state theory (CVT) with small-curvature tunneling (SCT) contribution has been successfully performed for formation of CPRs from the complete series reactions of 19 CP congeners with H and OH radicals ,35, and l values ,35. To b−12, 1.35 × 10−12, 1.49 × 10−12, 1.63 × 10−12, 1.68 × 10−12 cm3·molecule−1·s−1 for reactions of 2,3,4-TCP, 2,3,5-TCP, 2,3,6-TCP, 2,4,5-TCP and 2,4,6-TCP with H, while the value is 3.82 × 10−12 cm3·molecule−1·s−1 for that from 3,4,5-TCP with H. Similarly, at 1000 K, the CVT/SCT rate constants are 2.99 × 10−15, 1.38 × 10−15, 2.76 × 10−15, 2.44 × 10−15, 1.20 × 10−16 cm3·molecule−1·s−1 for reactions of 2,3,4-TCP, 2,3,5-TCP, 2,3,6-TCP, 2,4,5-TCP and 2,4,6-TCP with OH, while the value is 3.44 × 10−14 cm3·molecule−1·s−1 for that from 3,4,5-TCP with OH. This perfectly matches the structural and thermodynamic analysis above that the chlorine substitution at the ortho-position of CTPs increases the strength of the S–H bonds and decreases its reactivity.The chlorine substitution pattern of thiophenol strongly affects the CVT/SCT rate constants. At a given temperature, the calculated CVT/SCT rate constants for the thiophenoxyl-hydrogen abstraction from 2-CTP by H or OH radical is smaller than those of the thiophenoxyl-hydrogen abstraction from 3-CTP and 4-CTP by H or OH radical, respectively. The calculated CVT/SCT rate constants for the thiophenoxyl-hydrogen abstraction from 2,3-DCTP, 2,4-DCTP, 2,5-DCTP and 2,6-DCTP by H or OH radical are smaller than those of the thiophenoxyl-hydrogen abstraction from 3,4-DCTP and 3,5-DCTP by H or OH radical, respectively. The CVT/SCT rate constants for the thiophenoxyl-hydrogen abstraction from 2,3,4-TCTP, 2,3,5-TCTP, 2,3,6-TCTP, 2,4,5-TCTP and 2,4,6-TCTP by H or OH are smaller than that of the thiophenoxyl-hydrogen abstraction from 3,4,5-TCTP by H or OH, respectively. For example, at 1000 K, the CVT/SCT rate constants are 2.54 × 10−12 cm3·molecule·s−1, whereas the value is 2.91 × 10−15 cm3·molecule·s−1 for the thiophenoxyl-hydrogen abstraction from 2,3-DCTP by OH. This is consistent with thermodynamic analysis: Thiophenoxyl-hydrogen abstraction from CTPs by H is more efficient than the thiophenoxyl-hydrogen abstraction by OH.For a given thiochlorophenol, the CVT/SCT rate constants for the thiophenoxyl-hydrogen abstraction by H are noticeably larger than those of the thiophenoxyl-hydrogen abstraction by OH over the whole studied temperature range. For example, at 1000 K, the CVT/SCT rate constant of the thiophenoxyl-hydrogen abstraction from 2,3-DCTP by H is 2.66 × 10Comparison with the previous studies of phenoxyl-hydrogen abstraction by H and OH from CPs shows that the CVT/SCT rate constant for the reaction of CTP with H is consistently larger than that of corresponding CP with H at a given temperature , whereasThe Gaussian 09 program  was usedRate constants in this study over a wide temperature range (600–200 K) were calculated using the canonical variational transition state theory (CVT) with small-curvature tunneling (SCT) correction ,42,43,44The optimized geometries of thiophenol and the calculated vibrational frequencies of thiophenol and 4-chlorotriophenol at the MPWB1K/6-31+G level are consistent with the available experimental values, and the relative deviation remains within 1.0% for the geometry parameters and 9.0% for the vibrational frequencies ,46,47. TIn this study, we investigated the theoretical formation of chlorothiophenoxy radicals (CTPRs) from the complete series reactions of 19 chlorothiophenol (CTP) congeners with H and OH radicals using DFT electronic structure theory and canonical variational transition-state (CVT) theory with the small curvature tunneling (SCT) contribution. Structural parameters were calculated for all the stationary . Potential barriers, reaction heats, and rate constants for all the elementary reactions were studied to compare the formation potential of CTPRs from CTPs with H and OH radicals. Comparison of this study with our previous studies of the chlorophenoxy radical (CPR) formation from chlorophenols (CPs) with H and OH radicals were discussed ,35. Threortho chlorine increases the strength of the S–H bond in CTPs and decreased its reactivity, i.e., decreases the formation potential of CTPRs from the ortho-substitued CTPs with H and OH radicals.(1) The (2) The triophenoxyl-hydrogen abstraction from CTPs by H is more efficient than the phenoxyl-hydrogen abstraction from CPs by H, whereas the thiophenoxyl-hydrogen abstraction from CTPs by OH is less impactful than the phenoxyl-hydrogen abstraction from CPs by OH.(3) Different from reactions of CPs with H and OH, the thiophenoxyl-hydrogen abstraction from CTPs by H can occur more readily than the thiophenoxyl-hydrogen abstraction by OH radical.The obtained results can support the important input parameters for the PCDT/TA control models in the environment, and be used for future estimates of PCDT/TAs emission quantity based on the well estimated PCDT/TA inventory.